NL2033301B1 - Dominant epitope fusion protein of african swine fever virus (asfv) non-structural protein and kit and use thereof - Google Patents
Dominant epitope fusion protein of african swine fever virus (asfv) non-structural protein and kit and use thereof Download PDFInfo
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Abstract
The present disclosure belongs to the technical field of genetic engineering and provides an African swine fever virus (ASFV) non— structural protein dominant epitope fusion protein and a kit and use thereof. The dominant epitope fusion protein of an ASFV non— structural protein has an amino acid sequence shown in SEQ ID NO. l. The fusion protein has good immunoreactivity with African swine fever virus positive serum. The obtained dominant epitope fusion protein of an ASFV non—structural protein is used as a coating antigen to establish an ELISA. method, for detecting an African swine fever virus non—structural protein antibody. The detection kit has the characteristics of strong specificity, high sensitivity, good repeatability, and high accuracy, can effectively detect antibodies produced after African swine fever virus infection, and provides important assistance for research on diagnosis and infection mechanism. of African swine fever and development of safe and efficient vaccines.
Description
DOMINANT EPITOPE FUSION PROTEIN OF AFRICAN SWINE FEVER VIRUS
(ASFV) NON-STRUCTURAL PROTEIN AND KIT AND USE THEREOF
The present disclosure belongs to the technical field of ge- netic engineering and particularly relates to a dominant epitope fusion protein of an African swine fever virus (ASFV) non- structural protein and a kit and use thereof.
African swine fever (ASF) is a highly contagious disease of pigs caused by African swine fever virus (ASFV), has clinical symptoms and pathological changes similar to classical swine fe- ver, and has relatively high morbidity and mortality. After intro- duced into China from 2018, ASF brings huge loss to the live pig breeding industry of China. Although strict prevention and control measures are taken, ASF is still popular and outbreaks in related areas, and seriously affects stable and healthy development of the pig breeding industry of China. ASFV is a linear double-strand DNA virus. A mature virion exhibits an icosahedral symmetry and has an envelope. A size of a genome has certain difference among differ- ent strains and is generally 170-190 kb. The genome contains more than 150 open reading frames and encodes more than 100 non- structural proteins and 68 structural proteins. The non-structural protein plays an important role in replicating and assembling vi- ruses, regulating host cell functions, and escaping host natural immunity and the like. However, there are relatively few methods for detecting ASFV non-structural protein antibodies, and most of clinical applications are for detecting ASFV structural protein antibodies, which seriously restricts in-depth research and under- standing of a pathogenic mechanism of ASFV.
A238L protein is an important non-structural protein of ASFV, can regulate related biological functions of host cells during an infection process of the virus, and thus creates a good living space for ASFV. Since the protein contains a 1 kb ankyrin repeat,
it is considered to be a 1 kb analog. In different infected cells, the molecular weight of the protein is slightly different due to influence of different intracellular environments. A study of
Silk, et al. shows that A238L protein can exist in cytoplasm and cell nucleus simultaneously, is accumulated in the cell nucleus 10-18 h after infection, can inhibit activation of NF-kappa B, and helps ASFV to escape a natural immune reaction of a host. In addi- tion, A238L protein can also inhibit activation of transcription factors related to nuclear factor of activated T-cells, such that related immune functions of host cells are lost and replication and transcription of ASFV virions are promoted. A238L protein can inhibit transcription of an inducible nitric oxide synthase gene by inhibiting acetylation of p65 and activity of p300 in macro- phages, thereby reducing generation of an inflammatory mediator of nitric oxide and resisting inflammation. However, in a research process of an A238L protein molecule-related action mechanism, an
ASFV non-structural protein A238L whole gene sequence is mostly subjected to a prokaryotic expression and an eukaryotic expres- sion, such that the expressed sequence contains sequences which have no interaction with host proteins, and thus the interaction between A238L protein molecules and target molecule sequences is seriously affected, and interaction sites between the A238L pro- tein molecules and the target protein molecules cannot be accu- rately displayed. In addition, containing non-acting sequences may affect an expression level of a target recombinant protein or cause non-expression of the target recombinant protein. The tech- nology of the project carries out a tandem expression on dominant epitopes of ASFV non-structural protein A238L and eliminates non- related sequences, which can fully present the dominant epitope of the protein and improves the binding efficiency of the protein and an antibody of ASFV. When the ASFV non-structural protein A238L is used as a coating antigen, an ASFV non-structural protein antibody can be accurately detected to determine epidemic dynamic of ASFV in a swine herd and an infection replication rule of ASFV in a swine body, which provides important assistance for research on diagnosis and infection mechanism of ASF and development of safe and efficient vaccines.
In view of this, the present disclosure aims to provide a dominant epitope fusion protein of an African swine fever virus (ASFV) non-structural protein and a kit and use thereof. Four dom- inant epitopes of ASFV non-structural protein A238L protein are in tandem to obtain the dominant epitope fusion protein of an ASFV non-structural protein and the fusion protein has good immunoreac- tivity with ASFV positive serum. The kit uses the dominant epitope fusion protein of an ASFV non-structural protein as a coating an- tigen and has characteristics of good specificity, sensitivity, repeatability, and accuracy when detecting whether pig serum con- tains an ASFV antibody.
To achieve the above objective, the present disclosure pro- vides the following technical solutions.
The present disclosure provides a dominant epitope fusion protein of an African swine fever virus non-structural protein, having an amino acid sequence shown in SEQ ID NO. 1.
The present disclosure provides a nucleotide encoding the dominant epitope fusion protein of an African swine fever virus non-structural protein.
Preferably, the nucleotide sequence may be shown in SEQ ID
NO. 2.
The present disclosure provides an expression vector contain- ing the nucleotide.
The present disclosure provides a host bacterium containing the nucleotide or the expression vector.
The present disclosure provides an ELISA antibody detection kit for a dominant epitope fusion protein of an African swine fe- ver virus non-structural protein, where the kit includes an ELISA plate and the ELISA plate is coated with the dominant epitope fu- sion protein of an African swine fever virus non-structural pro- tein.
Preferably, the kit may further include a coating buffer, a washing buffer, and a blocking buffer.
The present disclosure provides a method for preparing a dom- inant epitope fusion protein of an African swine fever virus non-
structural protein, comprising the following steps: constructing the expression vector, transforming the expres- sion vector into a host bacterium, after induced expression of the host cell containing a dominant epitope fusion protein of an Afri- can swine fever virus non-structural protein, performing ultrason- ication and inclusion body dissolution and purification, and col- lecting the dominant epitope fusion protein of an African swine fever virus non-structural protein.
The present disclosure provides a method for preparing an Af- rican swine fever virus non-structural protein antibody, compris- ing the following steps: immunizing an animal with the fusion protein as an antigen to produce the African swine fever virus non-structural protein anti- body in an animal.
The present disclosure provides use of the fusion protein, the nucleotide, the expression vector, or the host bacterium in preparing a product for detecting an African swine fever virus.
Compared with the prior art, the present disclosure has the following beneficial effects: the present disclosure provides a dominant epitope fusion protein of an African swine fever virus non-structural protein and a kit and use thereof. Four dominant epitopes of ASFV non- structural protein A238L protein are in tandem to obtain the domi- nant epitope fusion protein of an ASFV non-structural protein and the fusion protein has good immunoreactivity with African swine fever virus positive serum. The kit uses a dominant epitope fusion protein of an ASFV non-structural protein as a coating antigen.
The established antibody detection kit has the characteristics of strong specificity, high sensitivity, good repeatability, and high accuracy, can effectively detect antibodies produced after African swine fever virus infection to determine epidemic dynamic of ASFV in a swine herd and an infection replication rule of ASFV in a swine body, and provides important assistance for research on di- agnosis and infection mechanism of African swine fever and devel- opment of safe and efficient vaccines.
FIG. 1 shows identification of amplification of a 4-dominant epitope tandem fusion protein of African swine fever virus non- structural protein A238L, vector construction, and expression 5 strain construction, wherein M: DL2000 DNA Marker, lane 1 is a PCR amplification result of an A238L-B gene sequence, lane 2 is a con- struction result of a Pet28a-A238L-B expression vector, and lane 3 is an identification result of E.Coli BL21/Pet28a-A238L-B bacteri- al liquid;
FIG. 2 is an SDS-PAGE electrophoresis identification after induced expression and purification of a 4-dominant epitope fusion protein of African swine fever virus non-structural protein A238L, wherein M: protein Marker and lane 1 is the expressed and purified 4-dominant epitope fusion protein of African swine fever virus non-structural protein A238L; and
FIG. 3 is identification of reactivity of a 4-dominant epitope fusion protein of African swine fever virus non-structural protein A238L, wherein M: protein Marker and lane 1 is the ex- pressed and purified 4-dominant epitope fusion protein of African swine fever virus non-structural protein A238L.
In order to fully present dominant epitopes of African swine fever virus non-structural protein A238L and improve specificity, sensitivity, and accuracy of the protein antigen in detection, a high-titer antibody of the protein is prepared, a whole gene se- quence of the African swine fever virus non-structural protein
A238L is analyzed, it is found that 4 dominant epitopes located in
A238L protein have better immunoreactivity and are highly con- served among different genotypes of an African swine fever virus.
The 4 dominant epitopes of an African swine fever virus non- structural protein A238L are subjected to tandem synthesis. There- fore, the present disclosure provides a dominant epitope fusion protein of an African swine fever virus non-structural protein, having an amino acid sequence shown in SEQ ID NO. 1.
The present disclosure further provides a nucleotide encoding the dominant epitope fusion protein of an African swine fever vi-
rus non-structural protein. Preferably, the nucleotide sequence may be as shown in SEQ ID NO. 2.
The present disclosure further provides an expression vector containing the nucleotide. In the present disclosure, preferably, a basic vector of the expression vector may be a Pet28a vector.
The present disclosure provides a host bacterium containing the nucleotide or the expression vector. In the present disclo- sure, preferably, a starting strain of the host bacterium may be
Escherichia coli BL21 (DE3). The source of the Escherichia coli
BL21 (DE3) is not particularly limited and a commercially availa- ble product in the art can be used.
The present disclosure provides an ELISA antibody detection kit for a dominant epitope fusion protein of an African swine fe- ver virus non-structural protein, where the kit includes an ELISA plate and the ELISA plate is coated with the dominant epitope fu- sion protein of an African swine fever virus non-structural pro- tein.
In the present disclosure, the kit may further preferably in- clude a coating buffer, a washing buffer, and a blocking buffer.
The coating buffer may preferably be 0.03-0.06 mol/L of a car- bonate buffer and the pH may preferably be 9.0-10.0. The washing buffer is a PBST solution and the PBST solution is a conventional reagent. The source of the PBST solution is not particularly lim- ited in the present disclosure, and the PBST solution can be pre- pared by oneself or purchased. The blocking buffer may preferably be 0.005-0.015 g/mL of BSA. In the present disclosure, a standard for determining results of a detection method of the kit is as follows: when an average value (N) of ODssonm Of a negative control is less than 0.25 and an average value (P) of ODggm Of a positive control is greater than 0.65, a sample is determined to be posi- tive when ODssom value (S) of the sample/the average value (N) of
ODssonm Of a negative control 2 2.1 and is determined to be nega- tive when ODssoan value (8S) of the sample/the average value (N) of
ODss9m Of a negative control < 2.1.
The kit of the present disclosure has the characteristics of strong specificity, high sensitivity, good repeatability, and high accuracy, and can effectively detect antibodies produced after Af-
rican swine fever virus infection to determine whether a pig is infected with an African swine fever virus.
The present disclosure provides a method for preparing a dom- inant epitope fusion protein of an African swine fever virus non- structural protein, comprising the following steps: constructing the expression vector, transforming the expres- sion vector into a host bacterium, after induced expression of the host cell containing a dominant epitope fusion protein of an Afri- can swine fever virus non-structural protein, performing ultrason- ication and inclusion body dissolution and purification, and col- lecting the dominant epitope fusion protein of an African swine fever virus non-structural protein.
In the present disclosure, the expression vector is con- structed and a basic vector of the expression vector may prefera- bly be a Pet28a vector. A nucleotide encoding the dominant epitope fusion protein of an African swine fever virus non-structural pro- tein is named an A238L-B gene sequence which is ligated between
BamH 1 and Xho 1 restriction sites in the Pet28a vector to obtain
Pet28a-A238L-B.
In the present disclosure, after the expression vector is constructed, the expression vector is transformed into a host bac- terium, where the transformation method may preferably be heat stress transformation and a starting strain of the host bacterium may preferably be Escherichia coli BL21 (DE3), that is, E. Coli
BL21/pET28a-A238L-B.
In the present disclosure, after induced expression of the host cell containing a dominant epitope fusion protein of an Afri- can swine fever virus non-structural protein, ultrasonication and inclusion body dissolution and purification are performed, and the dominant epitope fusion protein of an African swine fever virus non-structural protein is collected. The induced expression may preferably be induced by a final concentration of 0.5-1.5 mmol/L of IPTG. The ultrasonication may preferably be performed at a pow- er of 350-450 W for 3-7 s and after an interval time of 4-6 s, ul- trasonic treatment is performed for 25-35 min. The purification may preferably include His-Bind resin purification and dialysis.
When the His-Bind resin purification is performed, washing is per-
formed with a rinsing solution C and a rinsing solution D, and eluting is performed with an eluent in sequence. The rinsing solu- tion C may preferably be 7-9 mol/ L of urea, 0.05-0.15 mol/L of
NaH;P0;, and 0.005-0.015 mol/L of Tris-CL at a pH of 6.3. The rins- ing solution D may preferably be 7-9 mol/L of urea, 0.05-0.15 mol/L of NaH;PC,, and 0.005-0.015 mol/L of Tris-cl at a pH of 5.9.
The eluent may preferably be 7-9 mol/L of urea, 0.05-0.15 mol/L of
NaH,PO;, and 0.005-0.015 mol/L of Tris-cl at a pH of 4.5. The domi- nant epitope fusion protein of an African swine fever virus non- structural protein prepared by the present disclosure has a purity as high as 95% and has good immunoreactivity with African swine fever positive serum.
The present disclosure provides a method for preparing an Af- rican swine fever virus non-structural protein antibody, compris- ing the following steps: immunizing an animal with the fusion protein as an antigen to produce the African swine fever virus non-structural protein anti- body in an animal. The African swine fever virus non-structural protein antibody has a high titer and the antibody titer of a rab- bit serum antibody produced by the fusion protein is 1:128,000.
The present disclosure provides use of the fusion protein, the nucleotide, the expression vector, or the host bacterium in preparing a product for detecting an African swine fever virus. In the present disclosure, the product includes a kit or a reagent.
The technical solution provided by the present disclosure will be described in detail below with reference to examples, but they should not be construed as limiting the protection scope of the present disclosure.
Example 1 Preparation of dominant epitope fusion protein of
African swine fever virus non-structural protein (1) Screening and synthesis of gene sequence of dominant epitope fusion protein of African swine fever virus non-structural protein
In the example, a bioinformatics method was used to compre- hensively analyze hydrophilicity, antigenicity, and amino acid bi- osurface accessibility of a secondary structure of A238L protein of an African swine fever isolated strain Pig/HLJ/2018 (MK333180)
in GenBank, and 4 dominant epitope peptide segments were screened out, namely a 32-amino acid sequence of a 15-46th segment (N’-
IKKHIRNGNLTLFEEFFK
TDPWIVNRCDKNGS-C'’ , SEQ ID No. 3), a 36-amino acid sequence of a 65-100th segment (N’- FEQESYPGEIINPHRRDKDGNSALHYLAEKKNHLIL-C',
SEQ ID No. 4), a 16-amino acid sequence of a 144-159th segment (N’ -GADPTOKDYHRGFTAW-C’ , SEQ ID No. 5), and a 9-amino acid se- quence of a 195-203th segment (N’'-TGGLRKSPK-C’, SEQ ID No. 6) of an N’ end of A238L protein. A gene sequence corresponding to 32 amino acids in the 15-46th segment is (5'- at- taaaaaacatattagaaatgggaatcttacactatttgaggaattttttaaaacagatccgtg- gattgtcaatagatgcgataaaaatggatcc-3’, 96 bp, SEQ ID No. 7), a gene sequence corresponding to 36 amino acids in the 65-100th segment is (5'- tttgaacaagaatcttatcctggagaaataattaaccctcataggagggataaa- gatggaaactctgctttacattatttagctgagaaaaaaaatcatttaatcctg-3’, 108 bp,
SEQ ID No. 8), a gene sequence corresponding to 16 amino acids in 144-159th segment is (5'- ggagcagatccgactcaaaaagactatcatagaggttttactgcttgg-3’, 48 bp, SEQ ID
No. 9), and a gene sequence corresponding to 9 amino acids in 195- 203rd segment is (5’- accggtggtttaagaaaaagcccaaaa-3’, 27 bp, SEQ
ID No. 10) of the N'’ end. The gene sequences corresponding to the amino acid sequences of the 4 dominant epitopes were sent to San- gon Biotech (Shanghai) Co., Ltd. for tandem synthesis to obtain a dominant epitope fusion protein of an African swine fever virus non-structural protein.
The dominant epitope fusion protein of an African swine fever virus non-structural protein had an amino acid sequence shown be- low (SEQ ID NO. 1):
Nf -IKKHIRNGNLTLFEEFFKTDPWIVNRCDKNGSFEQESYPGEIINPHRRDKDGNSALHY
LAEKKNHLILGADPTQKDYHRGFTAWTGGLRKSPK-C’, a 93-amino acid sequence.
The dominant epitope fusion protein of an African swine fever virus non-structural protein had a nucleotide sequence shown below (SEQ ID NO. 2): 5’-— attaaaaacatattagaaatgggaatcttacactatttgag- gaattttttaaaacagatccgtggattgtcaatagatgcgataaaaatggatcctttgaacaa- gaatcttatcctggagaaataattaaccctcataggagggataaa- gatggaaactctgctttacattatttagctgagaaaaaaaatcatttaatcctgggag-
cagatccgactcaaaaagactatcatagaggttttactgcttggaccggtggtttaa- gaaaaagcccaaaa-3', 279 bp. (2) Construction of expression vector of dominant epitope fu- sion protein of African swine fever virus non-structural protein
The screened and synthesized gene sequence of a dominant epitope fusion protein of an African swine fever virus non- structural protein was named an A238L-B gene sequence, and primers
A238L-B-F and A238L-B-R were designed and sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis. Primer A238L-B-F is provided with an EcoRI restriction enzyme cutting site (underlined) and protective bases; primer A238L-B-R is provided with an Xho I re- striction enzyme cutting site (underlined) and protective bases; and PCR amplification was performed using the synthesized A238L-B gene sequence (279 bp) as a template.
A238L-BF: b'-cggaattcattaaaaaacatattagaaatg-3’ (SEQ ID
No.1l1l); and
A238L-BR: 5’-ggctcgagttttgggectttttcttaaac-3’/ (SEQ ID No.l12).
PCR amplification system for A238L-B gene sequence was 50 ul: 2 pL of the synthesized template, 10 uL of 5xPrimeSTAR Buffer, 1 pL of 100 umol/L of each of A238L-BF/R primers, 4 pL of 2.5 mmol/L of a dNTP mixture, 0.5 pL of 2.5 U/uL of PrimeSTAR HS DNA Polymer- ase, and 31.5 pL of ddH:0. PCR reaction conditions were as follows: denaturation at 95°C for 5 min, 30 cycles of 95°C for 50 s, 52°C for 20 s, and 72°C for 30 s, and extension at 72°C for 10 min. Af- ter the reaction ended, agarose gel at a concentration of 1% was used for identification and a specific target fragment of the
A238L-B gene sequence was purified and recovered (FIG. 1).
The purified and recovered A238L-B gene sequence was digested with EcoRI and XhoI, a plasmid vector Pet28a was digested with
EcoRI and XhoI, and the A238L-B sequence fragment was ligated with the plasmid vector Pet28a. The system was as follows: 4 pL of
A238L-B sequence (EcoRI-Xhol digestion), 4 uL of Pet28a (EcoRI-
XhoI digestion), 1 pL of a 10xligation buffer, and 1 pL of a T4
DNA ligase. The ligation was performed at 4°C for 16 h, the ligat- ed product was subjected to heat-stress transformation to BL21 (DE3) competent cells, the competent cells were spread on an LB plate containing kanamycin at a final concentration of 50 pg/mL,
culture was performed at 37°C for 16 h, a single colony was picked and inoculated into a LB liquid medium containing 50 pg/mL of kan- amycin, and plasmids were extracted for identification. A BL21 (DE3) strain (E.Coli BL21/Pet28a-A238L-B) containing the plasmid
Pet28a-A238L-B was obtained by PCR identification and screening (FIG. 1). (3) Expression, purification and reactivity of dominant epitope fusion protein of African swine fever virus non-structural protein
A single colony of E.Coli BL21/pET28a-A238L-B was picked and cultured overnight, the bacteria were transferred to a freshly prepared LB liquid medium containing kanamycin (50 pg/mL) at an inoculum size of 1%, culture was performed at 37°C and under shak- ing of 240 rpm for 2 h, when ODs am was 0.8-1.0, IPTG was added to a final concentration of 1.0 mmol/L, the bacteria were cultured at 37°C and under shaking of 240 rpm for 6 h, and the bacteria were collected by centrifugation. The obtained bacteria were resuspend- ed in PBS, lysozyme with a final concentration of 1 mg/mL was add- ed, the mixture was placed on ice for 30 min, the cells were dis- rupted by ultrasonication (ultrasonication at a power of 400 W for 5 s and after an interval time of 5 s, ultrasonic treatment for 30 min), centrifugation was performed at 10,000 rpm at 4°C for 15 min, the supernatant was discarded, and an inclusion body precipi- tate was obtained. The inclusion body was dissolved in binding buffer (8 mol/L of urea, 0.1 mol/L of NaH,PO,, and 0.01 mol/L of
Tris-CL at a pH of 8.0). After the inclusion body was dissolved, centrifugation was performed at 10,000 rpm at 4°C for 15 min, the supernatant was taken and equilibrated with His-Bind resin and the binding buffer for 1 h, the filtrate was discarded, and the resi- due was washed with rinsing solution C (8 mol/L of urea, 0.1 mol/L of NaH:PO,, and 0.01 mol/L of Tris-CL at a pH of 6.3) and filtered twice, and then washed with rinsing solution D (8 mol/L of urea, 0.1 mol/L of NaH,PO,, and 0.01 mol/L of Tris-CL at a pH of 5.9) and filtered 4 times, and a target protein was collected with an elu- ent (8 mol/L of urea, 0.1 mol/L of NaH;PO4, and 0.01 mol/L of Tris-
CL at a pH of 4.5). The recombinant protein purified by His-Bind resin was placed in a pretreated semipermeable membrane (boiled in water at 100°C for 10 min), a reduced glutathione at a final con- centration of 1 mg/mL was added, and dialysis was performed slowly at 4°C in PBS containing 4 mol/L, 3 mol/L, 2 mol/L, and 1 mol/L of urea at a pH of 8.5 separately. After the dialysis, the purified
A238L-B protein was collected, the protein concentration was de- termined, and the protein was identified by SDS-PAGE electrophore- sis.
The result was shown in FIG. 2 below. A specific band of a dominant epitope fusion protein of an African swine fever virus non-structural protein (about 33.0 kD) was successfully obtained.
The purity of the dominant epitope fusion protein of an African swine fever virus non-structural protein was 95%.
After SDS-PAGE electrophoresis, Western-Blot was performed to identify the immunogenicity of the protein. Nitrocellulose mem- brane (NC) and filter paper of the same size as a gel strip were pre-cut and soaked in an electroporation buffer. The gel was taken out, after the equilibration in the electroporation buffer, a mem- brane, a gel, and a filter paper were applied in order, i.e. fil- ter paper-NC membrane-gel-filter paper, and the interlayer was gently rolled with a clean glass rod to remove bubbles between layers. After a tight sealing, the sealed layers were put into an electroporation tank, an electroporation solution was added, a cooling device was connected, and electroporation was performed at a constant current of 200 mA for 1 h. After the electroporation, the NC membrane was taken out and washed with TBST for 5 minx3 times; the NC membrane was put in 5% skim milk and blocked over- night at 4°C, a blocking buffer was discarded, and the membrane was washed with TBST for 5 minx3 times; a primary antibody was added, that is, African swine fever antibody-positive pig serum was diluted with TBST (1:100), the mixture was shaken steadily at a room temperature for 1 h, the primary antibody was discarded, and the membrane was washed with TBST for 5 minx4 times; a horse- radish peroxidase-labeled rabbit anti-pig enzyme-conjugated sec- ondary antibody was added and diluted with TBST at a ratio of 1:50,000, the mixture was shaken steadily at a room temperature for 1 h, the secondary antibody was discarded, and the membrane was washed with TBST for 5 minx3 times. The NC membrane treated with the secondary antibody was placed in a chromogenic solution for color development. After the specific reaction band appeared, the reaction was stopped and a picture was taken for preservation.
The result of the Western-Blot analysis was shown in FIG. 3.
The dominant epitope fusion protein of an African swine fever vi- rus non-structural protein can be identified by African swine fe- ver virus positive serum and has good immuncreactivity with the
African swine fever virus positive serum.
Example 2 Use of ELISA antibody detection kit for dominant epitope fusion protein of African swine fever virus non-structural protein in serologic detection
The dominant epitope fusion protein of an African swine fever virus non-structural protein A238L prepared in example 1 was used to establish an indirect ELISA method for detecting an African swine fever virus non-structural protein antibody: the dominant epitope fusion protein of African swine fever virus non-structural protein A238L was diluted with a coating buffer (0.05 mol/L of a carbonate buffer at a pH of 9.6) to 2.0 ng/mL, the diluted protein was added into an ELISA plate at an amount of 100 uL/well, and the plate was coated overnight at 4°C; 300 pL of PBST was added to each well for washing twice, and the plate was patted dry and blocked with 100 pL/well of a blocking buffer (0.01 g/mL BSA) at 37°C for 2 h; 300 pL of PBST was added to each well for washing twice, the plate was patted dry, 100 pL of pig serum to be detected diluted at 1:100 was added to each well, two wells for African swine fever virus antibody positive control samples (100 uL/well) and two wells for negative control samples (100 uL/well) were set at the same time, incubation was performed at 37°C for 30 min, and the liquid in the wells was dis- carded; 300 pL of PBST was added to each well for washing for 5 times, the plate was patted dry, 100 pL of HRP-labeled rabbit an- ti-pig IgG diluted at 1:50, 000 was added to each well, reaction was performed at 37°C for 30 min, and the liquid in the well was discarded; 300 uL of PBST was added to each well for washing for 5 times, the plate was patted dry, 100 pL of a TMB single-component color developing solution was added to each well, and color devel- opment was performed in a dark place for 10 min; and 100 pL of 2 mol/L of H;30, was added to each well to stop color development and
ODa4sam value was measured with a microplate reader.
A standard for determining results of a detection method of the kit is as follows: when an average value (N) of ODssom of a negative control is less than 0.25 and an average value (P} of
OD4sonm Of a positive control is greater than 0.65, a sample is de- termined to be positive when OD: value (S) of the sample/the av- erage value (N)} of ODs4zenn of a negative control 2 2.1 and is de- termined to be negative when ODa4s9xm value (S) of the sample/the av- erage value (N) of OD of a negative control < 2.1. (1) Determination of specificity of detection method of kit
The detection method was used to detect a porcine blue-ear virus antibody, a porcine circovirus antibody, a classical swine fever virus antibody, a swine foot-and-mouth disease virus anti- body, a porcine pseudorabies virus antibody, a porcine encephali- tis B virus antibody, a porcine parvovirus antibody, a haemophilus parasuis antibody, and a swine streptococcus suis type 2 antibody- positive swine serum, and the result was shown in Table 1.
Table 1 Detection results of kit on common pig disease posi- tive serum
Swine foot-
Porcine Classical Porcine
Porcine and- Porcine Porcine Haemo- | Strepto-
Serum blue- swine ence- circo- mouth | pseudo- parvo- philus coccus types ear fever phalitis virus dis- rabies virus parasuis | suis type 2 virus virus B virus ease sE EE ESEPEE 0.211 0.210 0.201 0.198 0.207 0.215 0.197 0.206 0.204
Result leo
Note: An average value of ODssom Of a positive control in the test was 1.197 and an average value of ODssonm of a negative control is 0.214. When S/N 2 2.1, the result was positive and when S/N < 2.1, the result was negative. “-” indicated the result was nega- tive.
The results of table 1 showed that the detection result of the porcine blue-ear virus antibody, the porcine circovirus anti-
body, the classical swine fever virus antibody, the swine foot- and-mouth disease virus antibody, the porcine pseudorabies virus antibody, the porcine encephalitis B virus antibody, the porcine parvovirus antibody, the haemophilus parasuis antibody, and the swine streptococcus suis type 2 antibody-positive swine serum were all negative, indicating that the kit had a good specificity. (2) Determination of sensitivity of detection method
Six African swine fever antibody-positive pig serums were taken for dilution according to 1:50, 1:100, 1:200, 1:400, 1:800, and 1:1,600. The established indirect ELISA method for detecting an African swine fever virus non-structural protein antibody and an African swine fever antibody ELISA detection kit produced by
INGENASA (a Spanish company) were used for detection. The detec- tion results were shown in Table 2 and Table 3.
Table 2 Detection results of positive serums with different dilution ratios by kit of the example
D
1:50 1:100 1:200 1:400 1:800 1:1600 rer fre [ume Jue Jom ones [oane
Mes ame Juste lon ome ese om
Note: An average value of ODssom Of a positive control in the test was 1.298 and an average value of ODssonm of a negative control is 0.227. When S/N 2 2.1, the result was positive and when S/N < 2.1, the result was negative. “=” indicated the result was nega- tive.
Table 3 Detection results of positive serums with different dilution ratios by kit (INGENASA, Spanish) ae on jn en en 1:50 1:100 1:200 1:400 1:800 1:1 600
No. 1 0s:a(y | 11340) | 17190)
No. 2 08740) | 16140) 1.809 ()
Note: An average value of ODgsonn of a positive control in the test was 0.134 and an average value of ODsssan of a negative control is 1.817. A sample blocking rate (3) = (OD value of a negative control-0D value of a sample) / (OD value of a negative control-0D value of a positive control) *x100%. When the sample blocking rate (%) 2 50%, the result was positive. When the sample blocking rate (%) S 40%, the result was negative. “+” indicated the result was positive and “=” indicated the result was negative.
The results in Table 2 and Table 3 showed that the serum ti- ter of the kit of the present disclosure was 1:400-1:800, and the highest titer of the serum detected by the kit produced by IN-
GENASA (Spanish) was 1:50. Compared with the antibody titer of the kit produced by INGENASA (Spanish), the kit of the present disclo- sure for detecting African swine fever virus positive serum had significantly improved sensitivity, can more accurately monitor epidemic dynamics of an African swine fever virus in a swine herd, and provides important assistance for early diagnosis and detec- tion of an African swine fever virus. (3) Determination of repeatability of kit 3.1) Determination of repeatability within same batch of kit
The kit of the same batch was used to detect 30 pig serums of the known background, including 15 African swine fever antibody positive serums and 15 African swine fever antibody negative se- rums. Three replicates were performed on each of the 30 serum sam- ples.
Table 4 Results of 3 replicates of pig serum samples detected by kit of same batch
Detection method of same batch
No. of Standard Variable
Serum type Coated plate Coated plate Coated plate serum deviation coefficient 1 2 3 1 1.218 1.104 1.124 0.0608 5.30% 2 1.134 1.086 1.034 0.0500 4.61% 1.165 1.254 1.198 0.0449 3.73% 1.598 1.617 1.715 0.0627 3.82% 1.841 1.784 1.756 0.0433 2.42% 6 1.138 1.224 1.123 0.0545 4.69% 7 1.205 1.114 1.122 0.0503 4.39%
African swine fever virus 8 1.134 1.036 1.112 0.0514 4.70% positive serum 9 1.151 1.162 1,131 0.0157 1.37% 1.765 1.681 1.548 0.1094 6.57% 1.655 1.561 1.654 0.0539 3.33% 12 1.582 1.571 1.611 0.0206 1.30% 13 1.178 1.158 0.0225 1.91% 14 1.013 1.158 0.0765 6.96% 1.156 1.148 1.159 0.0056 0.49% 16 0.198 0.205 0.225 0.0140 6.69% 17 0.246 0.245 0.215 0.0176 7.48% 0.237 0.238 0.228 0.0055 2.35% 19 0.187 0.168 0.197 0.0147 8.01% 0.215 0.232 0.205 0.0136 6.28% 21 0.237 0.221 0.224 0.0085 3.74% 22 0.189 0.197 0.189 0.0046
African swine fever virus 0.214 0.201 0.215 0.0078 3.71% negative serum 24 0.226 0.217 0.238 0.0105 4.64% 0.196 0.198 0.189 0.0047 2.43% 26 0.217 0.218 0.197 0.0118 5.62% 0.229 0.214 0.224 0.0076 3.43% 0.216 0.232 0.223 0.0080 3.58% 29 0.217 0.219 0.224 0.0036 1.63% 0.225 0.217 0.214 0.0056 2.60%
The results in Table 4 showed that the variable coefficient within the same batch was between 0.49% and 6.96%, indicating that 5 the kit had a good repeatability within the same batch. 3.2) Repeatability test between batches
The kit of the different batches was used to detect 30 pig serums of the known background, including 15 African swine fever antibody positive serums and 15 African swine fever antibody nega- tive serums. Each of the 30 pig serum samples was detected in 3 batches by the detection method at the same time.
Table 5 Results of pig serum detected by different batches of kit ee TE Standard Variable coef-
Serum type eee positive serum 9 rar] 1.256 5.14%
The results in Table 5 showed that the variable coefficient among different batches was between 0.37% and 8.84%, indicating that the kit had a good repeatability among different batches. (4) Comparison test of clinical application of similar kits
The above kit was compared with the kit produced by INGENASA (Spanish) and 100 clinical pig serums were detected.
The results showed that when the ELISA antibody detection kit for a dominant epitope fusion protein of an African swine fever virus non-structural protein of the present disclosure was used to detect 100 serums, the positive rate was 48.00% (48/100) and the negative rate was 52.00% (52/100); and when the kit produced by
INGENASA (Spanish) was used for detection, the positive rate was 41.00% (41/100) and the negative rate was 59.00% (59/100); and a positive coincidence rate of the both kits was 85.42% (41/48), a negative coincidence rate was 88.14% (52/59), and an overall coin- cidence rate was 93.00% (93/100).
Example 3 Preparation of high-titer African swine fever virus non-structural protein antibody
Three healthy and clean-grade female New Zealand white rab- bits weighing 1.5-2 kg were purchased from Guangdong Medical La- boratory Animal Center. Two of the rabbits were selected to be im- munized with an expressed and purified dominant epitope fusion protein of African swine fever virus non-structural protein A238L, and the other rabbit was served as a negative control. The immun- ization was performed by a subcutaneous multi-point injection on the back. During the first immunization, the expressed and puri- fied epitope fusion protein of an African swine fever virus was mixed and emulsified with an equal amount of a Freund’s complete adjuvant and each rabbit in the immunization group was inoculated with 1 mL of the mixed agent with the content of the inoculated protein of 180 ug/rabbit. The rabbit in the negative control group was immunized with an emulsified mixed agent of PBS and a Freund's complete adjuvant at 1 mL/rabbit. During the subsequent immuniza- tion, the corresponding antigen was mixed and emulsified with a
Freund’s incomplete adjuvant. The immunization was performed with the same immunization dose and method. Each immunization interval was 15 days. On the 10th day after the third immunization, blood was collected to separate serum to determine antibody titer. An
ELISA plate (100 uL/well) was coated with the expressed and puri-
fied dominant epitope fusion protein of an African swine fever vi- rus (2.0 pg/mL) overnight at 4°C, washed with PBST for 3 times, and blocked with 0.01 g/mL of BSA at 100 uL/well. After the action at 37°C for 2 h, washing was performed 3 times with PBST, the se- rums of the three rabbits were sequentially diluted by PBS at a ratio of 1:1,000 and added at 100 pL/well, the action was per- formed at 37°C for 30 min, and washing was performed 3 times with
PBST; an HRP-labeled goat anti-rabbit enzyme-conjugated secondary antibody was diluted at 1:50,000 and added at 100 pL/well, the ac- tion was performed at 37°C for 30 min, and washing was performed 3 times with PBST; and a TMP single-component substrate was added at 100 pL/well, color development was performed at 37°C for 10 min, and the reaction was terminated with a 2 M sulfuric acid stop so- lution at 100 pL/well. The ODasonm value was read with a microplate reader.
Table 6 Determination of antibody titer of epitope fusion protein of African swine fever virus = Fe oom ooo
No. 1 immunized 2.926 2.614 2.206 1.841 1.213 1.071 0.865 0.561 0.214
No. 2 immunized 2.916 2.704 2.187 1.685 1.021 0.974 0.746 0.513 0.208 0.237 0.241 0.235 0.219 0.221 0.217 0.215 0.213 0.214
Control rabbit
The results in Table 6 showed that when the serums of the ex- perimental rabbits were diluted to 1:128,000, the ODssgnn value of the two immunized rabbits was still 2.1 times the ODsom value of the negative control rabbit. Therefore, the antibody titer of the rabbit serum immunized with the epitopes was 1:128,000.
The above descriptions are merely preferred implementations of the present disclosure. It should be noted that a person of or- dinary skill in the art may further make several improvements and modifications without departing from the principle of the present disclosure, but such improvements and modifications should be deemed as falling within the protection scope of the present dis- closure.
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HYLAEKKNHLILGADPTQKDYHRGFTAWTGGLRKSBK<//INSlSeg seuuence> 36 </INSDSeg> u </SequenceData> 41 <Hedgquencelata seguanoelhNumbag="2vs 42 <INSDSeq> az <INSDSeq length»278</INSDSeq length» 44 ZINSDSegq molitypes>DNAC/INSDSeq moltyper> 4% <IN3DSeq divisior>PAT</INSDIeqg division» 48 <INSDSeq feature-table> 47 <INSDFeature> 4d <INSDFeaturs keyrsource</INSDFeaturs Key» 4% <INSDFeature iccabion>l..278</INSDFaature location» 54 <INSDhFeature quels» a1 <INSDQualifier> 52 <IN3DQualifier name>mol type</INSDQualifisr name> 53 <INSDQualifiler valuerother DNA</INGDGualifier value» 54 </INSDQualifier>
LE <INSDQueiifier id="gs">
LE CINSDQualifisr name>note</INSDQvali fier name> 57 <INSDQualifier value>Nucleotide sequence of a dominant epitope fusion protein of ASFV</INSDQuelifier value» 58 </INSDQualifier> u 38 <INSDOQualifier id="g3">
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FO <INSDSeq> il <INSDSeq length>32</IN3SD3eq lengths vz <INSDSeq molityvpe>BAA</IN3DSeq moltype> 75 <INSDSeq division>PAT</INSDSeq divisicn>
TA <INSDSeq feature-iable> 75 <INSDPeature»>
Fi <IN3DFeature key>source</IiN3DFeature key» i <IN3DFeature lowation>l..32</INSDFeaturs location»
TH <INSDFeature guals> jd <INSDOualifier> 30 <INSDOQualifier name>mol type</INSDQualifier name>
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BE <{INSDQualifier» 37 <INSDQualifier id="g5">
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G4 <INSDSeq sequence>IKKHIRNGNLTLFEEFFKTDPWIVNRCDKNGS</IN3D3eq sequences
G5 </IN3SDSaq> 36 </SaquenceData> 37 “SequenceData seguencellNumber="47> 98 <INSDSeg> 38 <INSDSeg leng:b>36</INSDSeg length> 100 <INSDSeg moliype>AAc/INSDSeqg moltype>
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Lil <INSDQualifler id="gl%>
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Lin <INSDQualifier id=Vgiv>
Lig <INSDQualifier namerorganism</INSDQualiifier name>
LLY <INSDQualifier valuersynthetic construct“/INSDQualifien value»
LE </INSDOualifier> 113 </INSDFeature gualas> 120 </INSDFeature> oo
Lel </INSDSeg features table» 122 <IN3DSeqy sequence >FEQESYPGEIINPHRRDKDGNSALHYLAEKKNHLIL</INGDSeq sequenced
Las </INSDSeg>
Led </SequenceData>
Las <SequernceData sequenceliNuec="S"> 128 <INSDSeqg> 187 <INSDSeq length>l16</INSDSeq length> 128 <IN3DSeq moltyperAA</INSDSeq moltype> 12% ZINSDSeq division>PAT</INSDSeq division» 130 <INSDSeq feabure-table>
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TAD <INSDOQualifier namernote</INSDQualifiesr name> 14% <INSDQualiflisr value>A 16-amino acid sequence of a 144-159th segment /INSDGualifier value> 142 </INSDOualifier> 142 <INSDOualifier id="giin> 144 <IN3DQualifier name>note</INSDQualifier name> 145 <INSDQualifier valuerA 16-amino acid sequence of a 144-159th segment</INSDQualifier value» 146 «</INSDOQualifier»> 147 <INSDQualifler ia=’gS"> 148 <INSDQualifier name>organism</INSDQualifier name> 143 <IN3DQualifier value>synthetic construct /INSDQualifier values 150 </INSDOQuali fier u 151 </INSDFesature duals» 132 </INSDFeature> 15% </INSDSeg feature-tabhle> 134 <INSDSeq sequance>GADPTQKDYHRGFTAW<,/ INSD3ey sequence
LES </INSDSe 1548 </Seguencedata> isd <SequenceData semiencelDNunbern=N8#> i158 <INSDSeq> 15% <IN3DSeqy lengbh>9</INSDSeqg length>
Led <INSDSeq moliype>BAA</INSDSeq moltype»
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Led <INSDSeq feature-table> 163 <INZDFeature> 164d <INSDFeature key>sourcec/INSDFeature key» eh <IN3DFeature location>l..9</INSDFeature location» 188 <INSDFeature guals>
LET <INSDQualifier>
Las <INSDQualifier name>mol type</INSDQualiifier name>
LEG <INSDQualifier valuerprotein</IN3DQualifier value> 1G </INSDOualifiers jl <INSDQualiifler id="gZ8'>
LE <INSDQualifier name>note</INSDQualifier name> 17a <IN3DQualifier value>9-amino acid sequence of a 195-203th segment</INSDQualifier value>
Lid </INSDQuali fier»
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Lid <INSDQualiflsr value>synthetic construct /INSDQualifier value> 178 </INSDOualifier> 173 </IN3DFeature guala> ied </THSDFeatura> u
LEL </INSDSegy featurs-table>
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LEE ZINSDSegq moltype>DNA</INSDSeg moltyper>
LS <INSDSeag divisior>PAT</INSDIeqg division»
Led <INSDSeq feature-table>
Lal <INS3DFesature>
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Le <INSDQualifiler valuerother DNA</INGDGualifier value»
Les <{INSDQualifier»
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LAL </SequenceData>
ZAL “<SequenceData segusnceliNumec=NS8"> 242 <INSDSeqg> 242 <IN3DSeq length»48</INSDSeq length» 244 ZINSDSegq molitypes>DNAC/INSDSeq moltyper> 245 <IN3DSeq divisior>PAT</INSDIeqg division» 24% <INSDSeq feature-table>
ZA <INSDFeature> “4G <INSDFeaturs keyrsource</INSDFeaturs Key»
ZA <INSDFeature locabtiorn»l..48</INSDFeature location» 250 <INSDhFeature quels» 251 <INSDQualifier> 252 <IN3DQualifier name>mol type</INSDQualifisr name> 2573 <INSDQualifiler valuerother DNA</INGDGualifier value» 254 </INSDQualifier>» 205 <INSDQualifier ia=stglg¥>
PAS CINSDQualifisr name>note</INSDQvali fier name> 257 <INSDQualifier valus>Gene sequence corresponding to 16 amino acids /INSDQualifier valuer 258 </INSDQualifier> u 258 <INSDOualifier ld="gijn>
SAD <IN3DQualifier namerorganism“/INSDQuali fier name> 2a <INSDQualifier valuersynthetic construct</INSDQualifier valued a </INSDOualifiers 263 </INSDFeaturs guals> 264 </INSDFeaturer» 285 </INBDSeq feature-table>
LD
<INSDSeq sequence>ggagcagatccgactcaaaaagactatcatagaggttttactgettgg</INSDSe 4 sequence) 267 </INSDSeg>
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ZED <SequenceData seguanoellNumbeo="14%> <INSDSeqg> 271 <IN3DSeq length»27</INSDSeq length» 272 ZINSDSegq molitypes>DNAC/INSDSeq moltyper> 273 <INSDSeag divisior>PAT</INSDIeqg division» 274 <INSDSeq feature-table>
ZL <IN3DFeature> ais <INSDFeature keyvsource</INSDFeature key> zij <INSDFeature location>l..27</IN3DFeature location» 278 <INSDhFeature quels» 279 <INSDQualifier> 280 <IN3DQualifier name>mol type</INSDQualifisr name> 281 <INSDQualifiler valuerother DNA</INGDGualifier value» 282 </INSDQualifier> 283 <INSDQualifier ia=stgald¥> sd CINSDQualifisr name>note</INSDQvali fier name> zö5 <INSDQualifier value>Gene sequence corresponding to 9 amino acids</INsDOuzalifier valuer 288 </INSDQualifier> u 207 <INSDOualifier id="gisn> 288 <IN3DQualifier namerorganism“/INSDQuali fier name> 288 <INSDQualifier valuersynthetic construct</INSDQualifier valued
BGG </INSDOualifiers zel </INSDFeaturs duals? 252 </INSDFeaturer 233 </INBDSeq feature-table> 234 <IN3DSeqg sequencs>aceggtggtttaagaaaaagcccaaaa“/INSDSeg sequenced 285 </INSDSeg> 288 </Zequencelata> 297 <SequenceData zsegvencelnNumbser="iljx>
JARS <INSDieg>
Za “INSDSeq length>30</INSDSeq Length> 200 <INSDSeq moltype>DNA</INSDSeq moltype> 201 <IN3DSeq division»PAT</INSD3eq division» 302 <INSDSeq fearure-tabier 3203 <INSDFeabture> 304 <INSDFeature key>source</INIDFeature key> 305 <INSDFPeature locationrl..30</INSDFeature location 306 <INSDFeature qguals> u 307 <INSDQualifier»> 208 <INSDQualifier name>mol type</iNSDQualifier name> 203 <INSDuuelifier value>other DNA</INSDOualifier valuex zip </INSDQualifier> u
ZLD <INSDQualifier id='"g22"x> 34D <INSDQualifier namernote</INSDQualifier named
SLS <INSDQualifier valuerPrimer A238L-BF</INSDQualifier value»
SLá </INSDOualiLfier»> 315 <INSDQualiifler id="gZl"> 218 <INSDQualifier name>organism</iNSDQualifier name>
Lj <IiNSDgualifier value>synthetic construct</INSDQualifier valued» 3L8 </INSDQuali fier» 34e </INSDFearure quals>
SEU </INSDFeaturer
RY </INSDSeg feature-table>
Sl “INSDSeqg sequsnceveggaattcattaaaaaacatattagaaatg“/INSDSeq s=amquence>
Zeal </INSDSeg> 224 </SeguenceData> 225 <SeguenceData soquencelDNunmbey="12%"> 326 <INSDSeq» 327 <INSDSeq length>28</IN3SD3eq lengths
SES <INSDSeq moltype>DNA</INSDSegq moltype>
SEG <INSDSeq division>PAT</INSDSeq division» 330 <INSDSeq feature-iable>
SS <INSDPeature»> 222 <IN3DFeature keyrsource</INSDFeature key> 233 <IN3DFeature lowation>l..28</INSDFeaturs location» 334 <INSDFeature guals> 335 <INSDOualifier> 356 <INSDQualifier name>mol type</INSDQualifier name> 357 <INSDQualifler valverother DNA</INSDQualifier value» 338 </INSDOualifier> 333 <INSDOualifier id="q24%> 244 <IN3DQualifier name>note</INSDQualifier name> 241 <INSDQualifier value»Primer A238L-BR</INSDQualifi=sr valus> 342 </INSDOuali fier» u 343 <INSDQuaiifier id='"g23"x> 344 <INSDOQualifier namerorganism</INSDQualifier name> 345 <INSDQualiflsr value>synthetic construct /INSDQualifier value> 344 </INSDOualifier> 247 </IN3DFeature guala> 2498 </THSDFeatura> u 349 </INSDSegy featurs-table> 350 <INSDSeq seguencerggetegagttttgggetttttettaaac/INSDSeq sequence </INSDSeg>
ILE </SequenceData> 353 </8TZ2eSaguenceld sting
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