AU2021105170A4 - Attenuated Strain of African Swine Fever Virus without MGF360-9L Gene and Application thereof - Google Patents
Attenuated Strain of African Swine Fever Virus without MGF360-9L Gene and Application thereof Download PDFInfo
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Abstract
The present invention belongs to the field of biotechnology, and specifically relates to an
attenuated strain of African swine fever virus without MGF360-9L gene and its application.
The MGF360-9L gene of African swine fever virus has the function of significantly inhibiting
the host response. The attenuated strain of African swine fever virus without the MGF360-9L
gene adopts genetic engineering methods and uses homologous recombination from ASFV
CN/ GS/2018 isolate entire genome to knock out the gene MGF-360-9L to achieve the purpose
of weakening the virulence. Experiments with target animals in piglets show that the MGF360
9L monogene deletion attenuated strain of African swine fever virus of the present invention
has good safety in use and can be used as a candidate strain for preparation of African swine
fever vaccine.
1/2
MGF-360-9L P72 eGFP MGF-360-9L
Left Arrn Right Arm
pUC57
Figure 1
A Transmittent B GFP
Figure 2
Transmitint GFP Overlay
Figure 3
Description
1/2
MGF-360-9L P72 eGFP MGF-360-9L Left Arrn Right Arm
pUC57 Figure 1
A Transmittent B GFP
Figure 2 Transmitint GFP Overlay
Figure 3
Attenuated Strain of African Swine Fever Virus without MGF360-9L Gene and
Application thereof
The present invention belongs to the field of biotechnology, and specifically relates to an
attenuated strain of African swine fever virus without MGF360-9L gene and its application.
African swine fever (ASF) is a violent infectious disease of swine caused by African swine fever
virus (ASFV) infection. The disease has a short course of onset and has a case fatality rate close
to 100%. It has been listed as a legally reported animal disease by the Office International Des
Epizooties (OIE) and China also lists it as a type of animal disease. The disease was first
reported in Kenya in 1921 and was introduced to China in 2018. Subsequently, African swine
fever broke out in China and caused heavy losses to the pig industry. As of December 2019, it
had occurred in 32 provinces, cities, and autonomous regions. There were 161 outbreaks (157
domestic pigs and 4 wild boars), and over a million pigs were culled, making it the "top killer" of
pig industry in China. At present, the number of pigs in stock has dropped sharply and the price
of meat has soared, posing severe challenges to the healthy development of pig industry in
China, stable supply of pork, and stable economic development. Effectively responding to
African swine fever has become a common task faced by our country and even the international
community, and it is also the main battlefield demand in the current agricultural and rural areas.
Although scientists from various countries have worked hard to tackle key problems, researchers
have already had a deeper understanding of the biological knowledge of ASFV, but no major
breakthrough has been made in the commercialization of ASFV vaccines, and there are no
effective commercial ASFV vaccines available so far. According to the previous experience in
the prevention and control of African swine fever, once an African swine fever epidemic occurs,
it is generally eliminated through culling. This will not only lead to economic losses, but also
take a long time. As the most effective and economical means to prevent and control viral infectious diseases, vaccines are crucial to the prevention and treatment of African swine fever.
Therefore, there is an urgent need for innovations in safe and effective African swine fever
vaccines.
Innate immunity is the host's first line of defense against pathogen infection and invasion, and
plays an important role in the host's antiviral response. ASFV has evolved a variety of immune
escape mechanisms to antagonize natural immunity to ensure its own proliferation. This process
is generally performed by multiple virulence genes. This leads to the existence of multiple
virulence genes in natural ASFV inactivated vaccines, leading to greater toxic effect of vaccines
than the preventive effect. After vaccination, it is easy to cause the death of pigs. Therefore, the
research and development of an African swine fever vaccine that has both immunogenicity and
reduced toxicity and keeps pigs alive has been a research hotspot in recent years.
Live attenuated vaccines have the advantage of being able to effectively activate the body's
immune system. Live attenuated vaccines obtained by knocking out virulence genes can
maintain immunogenicity and have better protective effects, which is a hot spot of current
research. The Chinese patent CN110551695A provides a four-gene deletion attenuated strain of
African swine fever virus. The attenuated strain is a four-gene deletion attenuated strain of the
African swine fever virus SYl8 isolate. It lacks the functional proteins of the following genes:
CD2v gene encoding product and the coding products of the three multi-gene family genes
(MGF360 12L, MGF360 13L, MGF360 14L) were immunized 28 days after the piglets were
vaccinated, and then challenge test was carried out with the parental virus of ASFV. As a result,
the immunized pigs were completely protected. However, this attenuated strain will have a short
term rise in body temperature at the initial stage of vaccination, which poses a certain safety risk,
and the period for obtaining immunogenicity after vaccination is longer, requiring 28 days or
longer.
Up to now, virulence gene knockout vaccines still have the following problems: different strains
have different effects when the same gene is deleted, immune side reactions caused by insufficient deletion and excessive attenuation caused by multiple gene deletions will cause attenuated strains to lost immunogenicity or protection etc.; and there are many knock-out genes, complicated operations, and high costs, which can also lead to a decrease in the success rate of knock-out. Therefore, the selection of knockout virulence genes is also very important. It is often necessary to consider immune side reactions, and
It is necessary to take into account the safety.
In order to solve the problems above, the purpose of the present invention is to provide a
monogene deletion strain of African swine fever virus MGF-360-9L, which has good safety and
can be used as a strain for preparing African swine fever vaccine.
In order to achieve the objectives above, the present invention adopts the following technical
solutions:
In the first aspect, the present invention provides an attenuated strain of African swine fever
virus with the MGF360-9L gene deletion, and the attenuated strain is obtained by deleting the
MGF360-9L gene of the African swine fever virus.
As a preferred solution of the present invention, the African swine fever virus is a genotype II
African swine fever virus strain.
As a preferred solution of the present invention, the original strain (genotype II African swine
fever virus strain) is an African swine fever virus CN/GS/2018 isolate.
As a preferred solution of the present invention, the sequence of the deleted MGF360-9L gene is
shown in SEQ ID NO. 1.
The above-mentioned attenuated strain of African swine fever virus with the MGF360-9L gene
deletion is prepared by homologous recombination, and specifically includes the following steps:
(1) Construction of eGFP screening expression cassette: The p72 promoter sequence amplified
by PCR and the green fluorescent protein gene amplified by using the peGFP-N1 vector as a
template are connected by the method of fusion PCR.
(2) Construction of homologous recombination transfer vector: design the upstream and
downstream sequences of the MGF-360-9L gene as homologous recombination arms, clone
them into the backbone vector pUC57, and insert the p72-eGFP- SV40 polyA the gene
fragments of the screening expression cassette in the middle of gene sequences of the left and
right arms of the recombination transfer vector, and sequence to confirm the correctness of the
vector.
(3) Cell transfection and recombinant virus screening: the homologous recombination transfer
plasmid pUC57-LRAMGF-360-9L-eGFP was transfected into porcine BMDM cells, and after a
period of time, it was infected with the original strain of African swine fever virus, the cells
infected by the recombinant virus under a fluorescent microscope was observed. After cell
digestion, select a single fluorescent cell to repeatedly freeze and thaw, and then inoculate
BMDM cells again, and observe the cell wells with fluorescence.
Preferably, the original strain is an African swine fever virus CN/GS/2018 isolate.
(4) Purification and identification of recombinant virus: through limiting dilution, expansion of
culture, purity test, PCR determination of the target gene, to determine and obtain the purified
ASFV MGF-360-9L gene-deleted virus obtained.
The PCR assay of the target gene in the embodiment of the present invention shows that the
attenuated African swine fever virus strain lacks nucleotides 24164-25216 in the full-length
sequence of the original strain CN/GS/2018.
On the other hand, the present invention provides an attenuated strain of African swine fever
virus used in the preparation of an African swine fever vaccine applications.
In another aspect, the present invention provides a vaccine containing the above-mentioned
attenuated strain of African swine fever virus.
The present invention also provides primer pairs designed for the virulence gene MGF-360-9L,
the sequence of which is shown in SEQ ID NO: 2 to 3.
In addition, the present invention provides an application of the aforementioned primer pairs in
the preparation of a kit for identifying the aforementioned attenuated and original strains of
African swine fever virus with the MGF360-9L gene deletion.
In addition, the present invention provides a detection kit for identifying the above-mentioned
attenuated and original strain of African swine fever virus without MGF360-9L gene. The kit
includes the above-mentioned primer pairs.
The beneficial effects of the present invention are:
(1) The present invention obtains an attenuated African swine fever virus strain with a monogene
deletion of MGF-360-9L only by knocking out the ASFV virulence gene MGF-360-9L. This
strain has good safety and is suitable for preparation of the African swine fever vaccine and as
importantly practical significance for the preparation, prevention and control of African swine
fever vaccine.
(2) The MGF 360 family is one of the virulence genes that cause ASFV, and it itself contains
about 20 members. MGF 360-9L is one of the members of the MGF 360 family. The research of
the present invention has found that MGF 360-9L has the function of significantly inhibiting the
natural immune response of host cells, and the pathogenicity of ASFV is greatly reduced after
lack of MGF 360.
Figure 1 is a schematic diagram of CN/GS/2018 ASFV MGF-360-9L gene knockout strategy.
Figure 2 shows the situation of suspected recombinant virus-infected cells after 6 hours of
transfection with the homologous recombination transfer vector, followed by inoculation with
the AFSV CN/GS/2018 strain (MOI=1) and continuous culturing for 48 hours. The scale is
400PM. Panel A is a picture of cells in a bright field; Panel B is a picture of GFP fluorescence in
Panel A.
Figure 3 shows the fluorescence of a single GFP-positive cell inoculated with BMDM cells in a
96-well plate for 72 hours, with a scale of 400 [M.
Figure 4 is a diagram showing the purity identification results of recombinant virus AMGF-360
9L. WT is the ASFV CN/GS/2018 wild strain, and Deleted represents the AMGF-360-9L
recombinant strain.
Figure 5 shows the survival of the parent strain and the deletion strain after infecting animals.
Re-40 represents the AMGF-360-9L recombinant strain.
Figure 6 is a diagram showing the activity of MGF360-9L in inhibiting HT-DNA-induced
interferon. 1-70 represents the protein associated with African swine fever virus, and 40
represents MGF-360-9L.
The present invention will be described in detail below in conjunction with embodiments. The
following embodiments will help those skilled in the art to further understand the present
invention, but do not limit the present invention in any form. It should be pointed out that for
those of ordinary skill in the art, a number of adjustments and improvements can be made
without departing from the concept of the present invention. These all belong to the protection
scope of the present invention.
Biosafety license and African swine fever laboratory activity license: Lanzhou Veterinary
Research Institute of Chinese Academy of Agricultural Sciences is approved by the Ministry of
Agriculture with the help of report level by level made by Chinese Academy of Agricultural
Sciences Lanzhou The Biosafety Committee of the Institute of Veterinary Medicine, the
Laboratory Animal Ethics Committee, the Biosafety Committee of the Chinese Academy of
Agricultural Sciences, the Laboratory Animal Ethics Committee of the Lanzhou Veterinary
Research Institute of the Chinese Academy of Agricultural Sciences, and the Biosafety
Committee of the Lanzhou Institute of Veterinary Medicine, Chinese Academy of Agricultural
Sciences to carry out highly pathogenic ASFV pathogen and animal research, and have been
filed with the Ministry of Agriculture and Rural Affairs, which meets the requirements of
national biosafety level.
Sources of materials used in the embodiments:
Cells and viruses: The primary porcine alveolar macrophages (PAM) and bone marrow-derived
macrophages (BMDM) used in the present invention were all isolated from healthy pigs aged 2
to 4 months (purchased from Animal Center, Lanzhou Veterinary Research Institute, Chinese
Academy of Agricultural Sciences), after aseptically harvesting the cells, the red blood cells was
removed with red blood cell lysate (purchased from Biosharp). PAM and BMDM underwent
centrifugation at low speed; the supernatant was discarded, and the cells were resuspended in
RPMI 1640 containing 10% FBS; the cells were placed in culture medium (purchased from
Gibco); the culture medium was placed at 5% CO2 incubator at 37°C. For BMDM cell culture,
an additional 10 ng/mL final concentration of recombinant porcine GM-CSF (purchased from
R&D Systems) should be added to the RPMI 1640 complete medium and placed in a 37°C, 5%
C02 incubator for induction, every 2 to 3 days wash once; the non-adherent cells underwent
centrifugation and re-added to a new cell bottle with changed medium to continue induction, and
frozen or used after 3-7 days. PAM cells were used to amplify ASFV, and the virus titer was
determined. BMDM cells were used for plasmid transfection and virus recombination
experiments. ASFV CN/GS/2018 is a virus isolate obtained from the Lanzhou Institute of
Veterinary Medicine, Chinese Academy of Agricultural Sciences. It belongs to genotype II and
has a virus titer of 106 HAD5a. It is stored in aliquots at -80°C for later use.
Embodiment 1 Construction, purification and identification of MGF360-9L gene deletion
African swine fever virus
1.1 Construction of eGFP screening expression cassette
The p72 promoter sequence was obtained by PCR amplification, that is, from - 97 nt upstream
of the p72 gene to the sequence before the ATG start codon; meanwhile, using the peGFP-N1
vector as a template, the green fluorescent protein (enhanced green fluorescent protein, eGFP)
gene was obtained. The two genes were connected by fusion of PCR method to obtain the eGFP screening expression cassette gene fragment, named p72-eGFP-SV40 polyA, and the expression cassette sequence contained the SV40 polyA termination sequence.
1.2 Construction of homologous recombination transfer vector
The pUC57 vector was used as a backbone vector to construct a homologous recombination
transfer vector for MGF-360-9L gene knockout. The specific steps are as follows: design 1.5 kb
of the upstream and downstream sequences of MGF-360-9L gene as homologous recombination
arms and clone them into the backbone vector pUC57vector. In the left and right arm gene
sequences of the MGF-360-9L recombination transfer vector Insert p72-eGFP-SV40 polyA in
the middle to screen the expression cassette gene fragment. After the sequencing was correct, the
homologous recombination transfer vector was named pUC57-LRAMGF-360- 9L-eGFP;
plasmid DNA was extracted with an endotoxin-free plasmid extraction kit, and the concentration
was determined, and stored at -20°C for later use. Recombination strategy as shown in Figure 1,
the deleted MGF-360-9L gene is the nucleotide sequence located in 24164-25216 in the
complete gene sequence of the African swine fever virus and its nucleotide sequence is shown in
SEQ ID NO.1.
1.3 Cell transfection and recombinant virus screening
The homologous recombination transfer plasmid pUC-LR A MGF-360-9L-eGFP (2 g) was
transfected with 6 L JetPEI @ -Macrophage DNA transfection reagent into BMDM cells of pig
(the number of cells is about 106 cells/wells). After 6 hours of transfection, the complete culture
medium was discarded, and BMDM cells was directly infected with ASFV CN/GS/2018 purified
virus strain (MOI=1). After infection, the medium is not changed. After 48 hours, a fluorescence
microscope was used to observe the number of fluorescent cells and pictures were taken. A large
amount of fluorescent expression can be observed under the microscope, indicating that the
suspected recombinant virus successfully infects the cells (Figure 2). After the cells are digested,
all the fluorescent cells in the single wells were picked out, carefully blown away in a new petri
dish, settled for 1 hour; all the single fluorescent cells in the infected wells were picked out, and inoculated into the pre-paved 96-well cells plate after repeated freezing and thawing; observation of the cell wells with fluorescence once every 12 hours was carried out. After labeling, observation continued to 72 hours. The results showed that the proportion of the number of fluorescent cells in some wells could reach 100%, indicating that the recombinant virus was successfully constructed (Figure 3), and it was named ASFV AMGF-360-9L. Compared with the full-length sequence of the African swine fever isolate ASFV/CN/GS/2018, the recombinant strain lacks nucleotides 24164-25216.
1.4 Purification and identification of recombinant virus
100% positive wells were subjected to 10 times of limiting dilution and expansion culture to
obtain recombinant virus. During the period, the virus genome extraction kit (purchased from
Beijing Tiangen Biotechnology Co., Ltd.) was used to extract wild ASFV and recombinant
ASFV genomic DNA; the primer for ASFV MGF- 360-9L (ASFV MGF-360-9L-F:5'
TTGTCGATCGTTACGGACCC-3'; as shown in SEQ. ID NO:2, ASFV MGF-360-9L-R:5'
TTTGACTTTTCCTCCGGCGA-3', as shown in SEQ. ID NO: 3) was adopted to perform PCR
identification on its purity to confirm whether the deletion is successful. The results showed that
the MGF-360-9L gene has not been detected in the genome of the recombinant virus, as shown
in Figure 4. This result further shows that the recombinant virus has been successfully
recombined and has been purified.
Embodiment 2 titration of virus titer
The titration of African swine fever virus is performed by the 50% haemadsorption (HADa)
method. HAD5 test is carried out based on literature (Borca MV, Ramirez-Medina E, Silva E,
Vuono E, Rai A, Pruitt S, Holinka LG, Velazquez-Salinas L, Zhu J, GladueDP. Development of
a highly effective African swine fever virus vaccine by deletion of the I]77L gene results in
sterile immunity against the current epidemic Eurasia strain.JVirol. 2020. pii: JVI .02017-19)
and appropriately adjusted: primary PBMC was inoculated in a 96-well cell culture plate, and the
sample to be tested was diluted with 10-fold, and each well was inoculated with 0.02 ml. The virus infection can be judged by the rosette formed by the aggregation of red blood cells around the infected cells. Observation was performed for 7 days; according to the Reed and Muench method (Reed, L. and H. Muench, A The simple method of estimaing fifty percent endpoint.
American Journal of Epidemiology 1938.27: p.493-497) the half blood cell adsorbed dose was
calculated(HAD5o).
The measurement results showed that the HAD50 of the wild strain of African swine fever virus
was 105/100 microliters, and the HAD5oof the recombinant virus withoutthe MGF-360-9L gene
was 104.0/100 microliters, and the results showed that virus titer decreased after MGF-360-9L
gene deletion.
Embodiment 3 Animal challenge experiment
In order to detect the pathogenicity of the gene knock-out AMGF-360-9L strain, in this
embodiment, the pathogenicity of the piglets was evaluated by intramuscular injection with a
dose of 10 HAD5. Specifically, in this embodiment, 10 healthy Landrace piglets negative for the
African swine fever antigen antibody were divided into 2 groups, each of five piglets challenged
the parent strain (wild African swine fever virus strain) and the deleted strain respectively. After
that, the body temperature changes were measured every day, peripheral blood and saliva were
collected, and the blood content of ASFV virus was determined by the fluorescent quantitative
PCR method. The observation was terminated on the 19th day
Results: The pathogenicity evaluation showed that the parent strain showed typical symptoms of
ASFV after intramuscular injection of 10 HAD5o: high body temperature, and all died in the later
stage; while the deleted strain showed that most animals dropped to normal body temperature
after 5 days, and survived; only one animal showed typical symptoms of ASFV and died, as
shown in Figure 5. In conclusion, the pathogenicity of the MGF-360-9L deletion strain is
significantly different from that of the parental strain, and the virulence of AMGF-360-9L strain
withoutMGF-360-9L mostly weakened.
Emobodiment 4 MGF 360-9L significantly inhibits the natural immune response of host cells
The HEK293 cells in good condition (purchased from ATCC) were trypsinized and spread on a
24-well plate, placed at 37°C, 5% C02 in a cell incubator, cultured for 10 hours, and the cell
density was close to 70% to 80%. Lipofectamine TM 2000 (purchased from Invitrogen) can be
transfected. The plasmids used for transfection mainly include 100 ng of IFN--Luc reporter
plasmid (purchased from Lanzhou Ruibolai Biotechnology Co., Ltd.), and 10 ng of internal
ginseng Renilla luciferase reporter gene TK (purchased from Lanzhou Ruibolai Biotechnology
Co., Ltd.) and the MGF360-9L plasmid (according to the genome data of Georgia 2007/1
(FR682468.1, published by GenBank,). Taking the MGF 360-9L gene sequence in this strain as
a reference, synthesizing the gene and constructing a recombinant true nuclear expression
plasmid pEGFP-C1(K+)-MGF 360-9L, the gene was correctly inserted into the expression
plasmid by sequence determination) (100 ng), transfected with HT-DNA (purchased from
Sigma) (lg) 24 h followed by transfection (1 g/mL) for 12 h. At least three parallel holes are
required in the experiment to ensure the reliability of the experimental results. Add 50 [L
1 X passive lysis buffer to each well for 15-20 min at room temperature, and check the activity of
the dual luciferase reporter gene after full cleavage. The results are shown in Figure 6. Compared
with other African swine fever virus-related proteins, MGF360-9L can significantly inhibit the
activity of interferon induced by HT-DNA.
The above-mentioned embodiments are only preferred embodiments of the present invention,
which are only used to explain the present invention, but not to limit the implementation scope of
the present invention. Obviously, for those skilled in the art, other embodiments can be easily
made by means of replacement or change according to the technical contents disclosed in this
specification. Therefore, all changes and improvements made to the principles and conditions for
process of the present invention should be included in the patent application scope of the present
invention.
Claims (10)
1. An attenuated strain of African swine fever virus withoutMGF360-9L gene, which is obtained
by deleting MGF360-9L gene of African swine plague virus.
2. The attenuated strain of African classical swine fever virus withoutMGF360-9L gene
according to claim 1, which is characterized in that the African classical swine fever virus is a
type II African classical swine fever virus strain.
3. The attenuated strain of African classical swine fever virus withoutMGF360-9L gene
according to claim 1, which is characterized in that the African classical swine fever virus strain
with gene type II is an African classical swine fever virus CN/GS/2018 isolate.
4. The attenuated strain of African classical swine fever virus withoutMGF360-9L gene
according to claim 2, characterized in that the sequence of the deleted MGF360-9L gene is
shown in SEQ ID NO:1.
5. The method for preparing an attenuated strain of African swine fever virus withoutMGF360
9L gene according to claim 4, characterized in that the attenuated strain of African swine fever
virus withoutMGF360- 9L gene is obtained by deleting MGF360-9L gene in original African
swine fever virus by homologous recombination.
6. The method for preparing an attenuated strain of African classical swine fever virus
withoutMGF360-9L gene according to claim 5, characterized by comprising the following steps:
(1) Construction of eGFP screening expression cassette: The p72 promoter sequence amplified
by PCR and the green fluorescent protein gene amplified by using the peGFP-N1 vector as a
template are connected by the method of fusion PCR.
(2) Construction of homologous recombination transfer vector: design the upstream and
downstream sequences of the MGF-360-9L gene as homologous recombination arms, clone
them into the backbone vector pUC57, and insert the p72-eGFP- SV40 polyA the gene fragments
of the screening expression cassette in the middle of gene sequences of the left and right arms of
the recombination transfer vector
(3) Cell transfection and recombinant virus screening: the homologous recombination transfer
plasmid pUC57-LRAMGF-360-9L-eGFP was transfected into porcine BMDM cells, and it was
infected with the original strain of African swine fever virus; after cell digestion, select a single
fluorescent cell to repeatedly freeze and thaw, and then inoculate BMDM cells again;
Preferably, the original strain is an African swine fever virus CN/GS/2018 isolate.
(4) Purification and identification of recombinant virus: through limiting dilution, expansion of
culture, purity test, PCR determination of the target gene, to determine and obtain the purified
ASFV MGF-360-9L gene-deleted virus obtained.
7. Application of the attenuated strain of African classical swine fever virus withoutMGF360-9L
gene according to any one of claims 1 to 4 in preparing African swine plague vaccine.
8. A vaccine containing the attenuated strain of African classical swine fever virus deleted from
MGF360-9L gene according to any one of claims I to 4.
9. Application of the primer pairs shown in SEQ ID NO: 2-3 in preparing the kit for identifying
the attenuated strain of African classical swine fever virus and the original strain deleted from
MGF360-9L gene according to any one of claims 1-4.
10. A detection kit for identifying the attenuated strain of African classical swine fever virus
deleted from MGF360-9L gene according to any one of claims 1-4, characterized in that the kit
comprises primer pairs shown in SEQ ID NO: 2-3.
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