CN110628797A - Identification method of canine erythrocyte antigen DEA1.1 - Google Patents
Identification method of canine erythrocyte antigen DEA1.1 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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- C—CHEMISTRY; METALLURGY
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells
Abstract
The invention discloses an identification method of dog erythrocyte antigen DEA1.1, belonging to the technical field of genes, comprising the following steps: obtaining a dog erythrocyte surface antigen DEA1.1 gene, and carrying out codon optimization on a gene sequence; constructing a DEA1.1 expression vector, and carrying out gene synthesis on the modified DEA1.1 gene; transforming the synthesized DEA1.1 gene into DH5 alpha cells for amplification; transforming the amplified DEA1.1 gene into BL21 cells for mass expression; purifying DEA1.1 protein; mixing the DEA1.1 protein after recombinant expression and purification with dog blood, detecting the blood coagulation condition, and identifying the antigenicity of DEA 1.1. The invention recombines and expresses dog erythrocyte surface antigen DEA1.1 by means of genetic engineering, so as to achieve the purposes of quickly, simply and conveniently detecting the blood type of dog DEA1.1, improving the success probability of dog blood transfusion, reducing the medical cost of dogs and improving the survival rate of dogs after a large amount of blood loss.
Description
Technical Field
The invention relates to a method for determining DEA1.1, in particular to a method for identifying canine erythrocyte antigen DEA1.1, belonging to the technical field of genes.
Background
The dog erythrocyte blood type refers to a blood type system which is formed by a plurality of mutually related antigen antibodies and is a genetic character and comprises antigens capable of resisting specific antibodies on the surfaces of animal erythrocytes, and the blood type system is a blood type system, such as a DEA (DogErythrocyte antigen) system and a leukocyte antigen system of a dog. In non-immunized or mathematical animals, there are congenital alloantibodies in the plasma; other alloantibodies of acquired nature can be produced after transfusion sensitization. Hemolysis after transfusion or after eating breast milk in newborn puppies and cats may be caused by these alloantibodies. Alloantibodies can be tested by cross-matching tests and blood typing, which indicates the presence of alloantibodies.
There are at least 13 red blood cell blood types, but only 6 blood types are significant, and they are the DEA1.1, 1.2, 3, 4, 5, 7 blood types of the DEA system. The DEA1 blood group has multiple alleles, including DEA1.1, DEA1.2, DEA1.3 and a null blood group, DEA1.1 antigen being the most important, followed by DEA 1.2.
If the dog is DEA1.1 positive and the dog is DEA1.1 negative, the first transfusion will be sensitized and the second transfusion will soon cause transfusion hemolysis within 12 hours, resulting in disintegration of transfused red blood cells and even life-threatening. Therefore, a DEA 1.1-positive dog cannot be used as a blood-supplying dog for a DEA 1.1-negative dog. When a DEA1.1 positive male dog and a DEA1.1 female dog are mated, and a born DEA1.1 positive puppy eats breast milk, the puppy may have hemolytic disease, so that the puppy dies in a short time. Therefore, it is preferable not to mate DEA1.1 positive male dogs and DEA1.1 negative female dogs to prevent hemolysis of puppies after eating breast milk.
The dog blood types DEA1.1 and DEA1.2 are very important in blood transfusion, and the dog with DEA1.1 negative can be transfused to the dog with DEA1.1 negative, the dog with DEA1.1 positive and the dog with DEA1.2 positive, so the dog is also called as a general blood-supply dog or a universal blood-supply dog.
Clinically, when the dog which has not transfused blood is transfused for the first time, the cross matching test or the blood type test can not be carried out. However, if the first infusion of a compatible blood group is initiated, the half-life of the infused blood red cells is about 12 hours; if blood of a compatible blood group is first transfused, the half-life of the transfused blood erythrocytes is 21 days. Therefore, when the blood is transfused for the first time to dogs, the cross matching test is performed for better effect, and the blood transfusion is important according to the blood type or the test result. Clinically, it is not usually done in blood grouping or cross matching tests, and after the first blood transfusion, the dogs have better spirits, but the disease condition is more serious the next day, which may be the reason for the input of unmatched blood.
Therefore, there is a need for a rapid, simple and easy-to-popularize method for identifying canine erythrocyte antigen DEA1.1 with high sensitivity, which can prevent the above-mentioned problems in blood transfusion pairing.
Disclosure of Invention
The invention mainly aims to provide an identification method of dog erythrocyte antigen DEA1.1, which expresses the dog erythrocyte surface antigen DEA1.1 through gene engineering means in a recombination way so as to achieve the purposes of quickly, simply and conveniently detecting the blood type of the dog DEA1.1, improving the success probability of dog blood transfusion, reducing the medical cost of dogs and improving the survival rate of dogs after a large amount of blood loss.
The purpose of the invention can be achieved by adopting the following technical scheme:
the identification method of the canine erythrocyte antigen DEA1.1 comprises the following steps:
step (1), obtaining a dog erythrocyte surface antigen DEA1.1 gene, and performing codon optimization on a DEA1.1 gene sequence by using a gene engineering technology, wherein the sequence is SEQ ID NO. 1;
step (2), a DEA1.1 expression vector is constructed, two enzyme cutting sites are respectively added at two ends of a DEA1.1 gene after codon optimization, the NdeI sites are respectively at the 5 'end, the XhoI sites are at the 3' end, the sequence of the modified DEA1.1 gene is SEQ ID NO.2, and the modified DEA1.1 gene is subjected to gene synthesis and is connected to the corresponding sites of a pET30aCH1-BAB vector;
step (3), transforming the synthesized DEA1.1 gene into DH5 alpha cells for amplification, and extracting amplified plasmids;
step (4), the amplified DEA1.1 gene is transformed into BL21 cells for mass expression, and the supernatant is taken;
step (5), carrying out affinity chromatography purification on DEA1.1 protein, and determining the purity of the protein;
and (6) mixing the DEA1.1 protein obtained after recombinant expression and purification with dog blood, detecting the blood coagulation condition, and identifying the antigenicity of DEA 1.1.
Preferably, the method for transforming the synthesized DEA1.1 gene into DH5 α cells and amplifying the cells in step (3) and extracting the amplified plasmids is as follows: DH5 α electroporation competent cells were prepared, and then a vector containing the DEA1.1 gene was transformed into DH5 α electroporation competent cells.
Preferably, the steps for preparing the DH5 alpha electrotransformation competent cell are as follows:
diluting the concentration of DH5 alpha strain, and spreading the strain to LB solid culture medium for overnight culture; selecting a single colony grown after overnight culture, and adding the single colony into an LB liquid culture medium for overnight culture; adding overnight cultured bacterial liquid into sterile LB culture medium, culturing until the absorbance of bacterial liquid at OD600 is 0.5-0.8, taking out, cooling in ice water mixture, centrifuging, and collecting bacteria;
washing with pre-cooled ultrapure water and sterile glycerol for several times, discarding the supernatant, resuspending the thallus with glycerol and subpackaging on ice into EP tubes for storage.
Preferably, the step of transforming the vector containing the DEA1.1 gene into DH5 α electroporation competent cells is as follows:
adding a vector containing a DEA1.1 gene into a DH5 alpha electrotransformation competent cell, culturing on ice, and transferring the cell into an electroporation container for pulse; then adding LB culture medium into an electroporation container to culture cells, and coating the cells into a screening culture medium for overnight culture; and (4) picking overnight cultured colonies, adding the colonies into an LB screening culture medium for overnight culture, and then extracting amplified plasmids.
Preferably, in the step (4), the amplified DEA1.1 gene is transformed into BL21 cells for mass expression, and the supernatant is obtained as follows: preparing BL21 electrotransformation competent cells, transforming the amplified plasmid into BL21 competent cells, and coating the cells in an LB solid screening culture medium for overnight culture after transformation; taking a single colony for overnight culture, inoculating the single colony into an LB liquid culture medium, culturing until OD600 is 0.5-0.8, and adding IPTG for culture; centrifuging and collecting bacteria; and (3) ultrasonically crushing the thalli, centrifuging at low temperature, taking supernatant, and performing suction filtration by using a micron filter membrane for later use.
Preferably, the steps for preparing BL21 electroporation competent cells are as follows:
diluting BL21 strain concentration, spreading to LB solid culture medium for overnight culture; selecting a single colony grown after overnight culture, and adding the single colony into an LB liquid culture medium for overnight culture; adding overnight cultured bacterial liquid into sterile LB culture medium, culturing until the absorbance of bacterial liquid at OD600 is 0.5-0.8, taking out, cooling in ice water mixture, centrifuging, and collecting bacteria;
washing with pre-cooled ultrapure water and sterile glycerol for several times, discarding the supernatant, resuspending the thallus with glycerol and subpackaging on ice into EP tubes for storage.
Preferably, the transformation method for transforming the amplified plasmid into BL21 competent cells is as follows:
adding the amplified DEA1.1 gene into BL21 electrotransformation competent cells, and culturing on ice; transferring to an electroporation container for pulse; adding LB culture medium into an electroporation container to culture cells, and spreading the cells into a screening culture medium for overnight culture; overnight cultured colonies were picked and added to LB selection medium for overnight culture.
Preferably, the method for affinity chromatography purification of the DEA1.1 protein comprises the following steps: and (4) taking the cracked supernatant, passing through a His affinity chromatographic column, and collecting the purified protein.
Preferably, the method for determining purity comprises: and (4) carrying out SDS-PAGE electrophoresis on the purified protein, and judging the definition of protein bands according to the purity.
The invention has the beneficial technical effects that: the identification method of the dog erythrocyte antigen DEA1.1 provided by the invention has the advantages of high sensitivity and simple operation, can quickly detect the blood type of the dog DEA1.1, effectively prevents difficult pairing of dog blood transfusions due to different blood types, improves the success probability of the dog blood transfusions, reduces the medical cost of the dogs, improves the survival rate of the dogs after a large amount of blood loss, and also reduces the risk of losing favorite pets of people.
Drawings
FIG. 1 is a flowchart of a preferred embodiment of the method for identifying canine erythrocyte antigen DEA1.1 according to the present invention;
FIG. 2 is a schematic diagram of SDS-PAGE electrophoretic detection of DEA1.1 recombinant protein according to a preferred embodiment of the method for identifying canine erythrocyte antigen DEA1.1 of the present invention.
Detailed Description
In order to make the technical solutions of the present invention more clear and definite for those skilled in the art, the present invention is further described in detail below with reference to the examples and the accompanying drawings, but the embodiments of the present invention are not limited thereto.
As shown in fig. 1 and fig. 2, the identification method of canine erythrocyte antigen DEA1.1 provided in this example includes the following steps:
step (1), obtaining a dog erythrocyte surface antigen DEA1.1 gene, and performing codon optimization on a DEA1.1 gene sequence by using a gene engineering technology, wherein the sequence is SEQ ID NO. 1;
step (2), a DEA1.1 expression vector is constructed, two enzyme cutting sites are respectively added at two ends of a DEA1.1 gene after codon optimization, the NdeI sites are respectively at the 5 'end, the XhoI sites are at the 3' end, the sequence of the modified DEA1.1 gene is SEQ ID NO.2, and the modified DEA1.1 gene is subjected to gene synthesis and is connected to the corresponding sites of a pET30aCH1-BAB vector;
step (3), transforming the synthesized DEA1.1 gene into DH5 alpha cells for amplification, and extracting amplified plasmids;
step (4), the amplified DEA1.1 gene is transformed into BL21 cells for mass expression, and the supernatant is taken;
step (5), carrying out affinity chromatography purification on DEA1.1 protein, and determining the purity of the protein;
and (6) mixing the DEA1.1 protein obtained after recombinant expression and purification with dog blood, detecting the blood coagulation condition, and identifying the antigenicity of DEA 1.1.
Further, in this example, as shown in FIG. 1, the synthesized DEA1.1 gene was transformed into DH5 alpha cells and amplified in step (3), and the amplified plasmid was extracted as follows:
diluting DH5 alpha strain to appropriate concentration, spreading the diluted strain in LB solid culture medium, culturing at 37 ℃ overnight; picking single colony grown after overnight culture, adding the single colony into 5ml LB liquid culture medium, and culturing overnight at 37 ℃ and 200 rpm; adding the overnight cultured bacterial liquid into 500ml of sterile LB culture medium, culturing at 37 ℃ and 200rpm until the absorbance of the bacterial liquid at OD600 is 0.6, taking out the bacterial liquid from the container, cooling the bacterial liquid in an ice-water mixture for 30 minutes, centrifuging and collecting the bacterial liquid; washing with precooled ultrapure water for 2 times at 5000rpm for 15 min; washing with 10% sterile glycerol for 2 times at 5000rpm for 15 min; after the last washing, the supernatant was discarded, the cells were resuspended in 20ml of 10% glycerol and split-filled on ice into 1.5 ml EP tubes, 150 ul per tube, and stored at-80 ℃ for further use;
then, the vector containing the DEA1.1 gene was transformed into DH5 α electroporation competent cells by the following method: adding 2 ul vector containing DEA1.1 gene into DH5 alpha electrotransformation competent cell, and culturing on ice for about 5 min; transferred to a 2mm electroporation vessel and pulsed (200 ohm, 25uFd, 2.5 kv); immediately adding 300ul LB culture medium into an electroporation container, culturing cells at 37 ℃ for 40 minutes, spreading into a screening culture medium, and culturing at 37 ℃ overnight; selecting overnight cultured colonies, culturing at 37 deg.C in LB screening culture medium at 37 deg.C and 200rpm overnight; and extracting the amplified plasmid.
Further, in this example, as shown in FIG. 1, the DEA1.1 gene after amplification was transformed into BL21 cells in step (4) for mass expression, and the method for collecting the supernatant was as follows:
preparation of BL21 electroporation competent cells: taking BL21 strain to dilute to proper concentration, coating the strain into LB solid culture medium, culturing at 37 ℃ overnight; picking single colony grown after overnight culture, adding the single colony into 5ml LB liquid culture medium, and culturing overnight at 37 ℃ and 200 rpm; adding the overnight cultured bacterial liquid into 500ml of sterile LB culture medium, culturing at 37 ℃ and 200rpm until the absorbance of the bacterial liquid at OD600 is 0.6, taking out the bacterial liquid from the container, cooling the bacterial liquid in an ice-water mixture for 30 minutes, centrifuging and collecting the bacterial liquid; washing with precooled ultrapure water for 2 times at 5000rpm for 15 min; washing with 10% sterile glycerol for 2 times at 5000rpm for 15 min; after the last washing, the supernatant was discarded, the cells were resuspended in 20ml of 10% glycerol and split-filled on ice into 1.5 ml EP tubes, 150 ul per tube, and stored at-80 ℃ for further use;
the amplified plasmid is transformed into BL21 competent cells by the following method: 2 ul of the vector of the DEA1.1 gene after amplification is added into BL21 electrotransformation competent cells, and the cells are incubated for about 5 minutes on ice; transferred to a 2mm electroporation vessel and pulsed (200 ohm, 25uFd, 2.5 kv); immediately adding 300ul LB culture medium into an electroporation container, culturing cells at 37 ℃ for 40 minutes, spreading into a screening culture medium, and culturing at 37 ℃ overnight; selecting overnight cultured colonies, culturing at 37 deg.C in LB screening culture medium at 37 deg.C and 200rpm overnight;
coating the transformed cells in an LB solid screening culture medium, culturing at 37 ℃ overnight, taking a single colony, culturing at 37 ℃ and 200rpm overnight, inoculating the single colony into an LB liquid culture medium (kan +) according to the proportion of 1:100, culturing until the OD600 is about 0.6, adding IPTG (final concentration is 0.5 umol/L), culturing at 37 ℃ and 200rpm for 2 hours; centrifuging at 5000rpm for 20 min to collect bacteria; breaking the thalli by ultrasonic, centrifuging at low temperature, taking supernatant, and filtering with a 0.22 micron filter membrane for later use.
Further, in this example, as shown in FIG. 2, the method for affinity chromatography purification of DEA1.1 protein was: and (4) taking the supernatant cracked in the step (4), passing through a His affinity chromatographic column, collecting purified protein, and preliminarily judging the purity of the purified protein through SDS-PAGE electrophoresis, wherein the relative molecular mass of the protein determines the mobility of an SDS-protein compound in gel electrophoresis, and a detergent SDS in polyacrylamide gel has a large amount of negative charges, so that the charge amount of the protein can be ignored. Therefore, the moving speed and distance of the protein in the SDS gel electric field are completely dependent on the relative molecular mass (the speed is inversely proportional to the relative molecular mass) and are not influenced by the charge, the proteins with different relative molecular masses are separated in different sections of the gel, the detection result is shown in FIG. 2, the protein band is clear, the size is about 40 KD, and the size is consistent with the expected result.
In summary, the identification method of the dog erythrocyte antigen DEA1.1 provided in this embodiment recombines and expresses the dog erythrocyte surface antigen DEA1.1 by a genetic engineering means, so as to achieve the purposes of rapidly and simply detecting the blood type of the dog DEA1.1, improving the probability of successful dog blood transfusion, reducing the medical cost of dogs and improving the survival rate of dogs after a large amount of blood loss.
The above description is only for the purpose of illustrating the present invention and is not intended to limit the scope of the present invention, and any person skilled in the art can substitute or change the technical solution of the present invention and its conception within the scope of the present invention.
Sequence listing
<110> Guangzhou Yideno Biotechnology Ltd
<120> identification method of canine erythrocyte antigen DEA1.1
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1198
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atgaaatacc tattgcctac ggcagccgct ggattgttat tactcgcggc ccagccggcc 60
atggaagttc agctggttga atctggtggt gacctggtta aaccgggtgg ttctctgcgt 120
ctgtcttgcg ttgcttctgg tttcaacttc tctaactacg acatgaactg ggttcgtcag 180
gctccgggta aaggtctgca gtgggttgct tacatctctt ctggtggtat caacacctac 240
tacgctgacg ttgttcaggg tcgtttcacc atctctcgtg acaacgctaa atctatgctg 300
tacctgcaga tggaccgtct gcgtgttgaa gacaccgcta tgtactactg cgctggtgaa 360
ggtccgtact ctcgtggttc tggtctgttc ggttacggta tggactactg gggtccgggt 420
acctctctgt tcgtttcttc tggtggtggt ggttctggtg gtggtggttc tggtggtggt 480
ggttctgctc agccggttct gacccagccg ccgtctgttt ctggttctct ggaccagcgt 540
gttaccatct cttgcaccgg ttcttcttct aacgttggtt actcttcttc tgttggttgg 600
taccagcagt tcccgggtcg tggtccgcgt accatcatct acttcgacac ctctcgtccg 660
tctggtgttc cggaccgttt ctctggttct aaatctggta acaccgctac cctgaccatc 720
tctggtctgc gtaccgaaga cggtgctcac tactactgct cttcttggga ctctggtgtt 780
cgtgctccgg ttttcggttc tggtaccccg ctgaccgttc tgggttctgg atccgctgct 840
gaagctggta tcaccggtac ctggtacaac cagctgggtt ctaccttcat cgttaccgct 900
ggtgctgacg gtgctctgac cggtacctac gaatctgctg ttggtaacgc tgaatctcgt 960
tacgttctga ccggtcgtta cgactctgct ccggctaccg acggttctgg taccgctctg 1020
ggttggaccg ttgcttggaa aaacaactac cgtaacgctc actctgctac cacctggtct 1080
ggtcagtacg ttggtggtgc tgaagctcgt atcaacaccc agtggctgct gacctctggt 1140
accaccgaag ctaacgcttg gaaatctacc ctggttggtc acgacacctt caccaaag 1198
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<211> 1230
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catatgaaat acctattgcc tacggcagcc gctggattgt tattactcgc ggcccagccg 60
gccatggaag ttcagctggt tgaatctggt ggtgacctgg ttaaaccggg tggttctctg 120
cgtctgtctt gcgttgcttc tggtttcaac ttctctaact acgacatgaa ctgggttcgt 180
caggctccgg gtaaaggtct gcagtgggtt gcttacatct cttctggtgg tatcaacacc 240
tactacgctg acgttgttca gggtcgtttc accatctctc gtgacaacgc taaatctatg 300
ctgtacctgc agatggaccg tctgcgtgtt gaagacaccg ctatgtacta ctgcgctggt 360
gaaggtccgt actctcgtgg ttctggtctg ttcggttacg gtatggacta ctggggtccg 420
ggtacctctc tgttcgtttc ttctggtggt ggtggttctg gtggtggtgg ttctggtggt 480
ggtggttctg ctcagccggt tctgacccag ccgccgtctg tttctggttc tctggaccag 540
cgtgttacca tctcttgcac cggttcttct tctaacgttg gttactcttc ttctgttggt 600
tggtaccagc agttcccggg tcgtggtccg cgtaccatca tctacttcga cacctctcgt 660
ccgtctggtg ttccggaccg tttctctggt tctaaatctg gtaacaccgc taccctgacc 720
atctctggtc tgcgtaccga agacggtgct cactactact gctcttcttg ggactctggt 780
gttcgtgctc cggttttcgg ttctggtacc ccgctgaccg ttctgggttc tggatccgct 840
gctgaagctg gtatcaccgg tacctggtac aaccagctgg gttctacctt catcgttacc 900
gctggtgctg acggtgctct gaccggtacc tacgaatctg ctgttggtaa cgctgaatct 960
cgttacgttc tgaccggtcg ttacgactct gctccggcta ccgacggttc tggtaccgct 1020
ctgggttgga ccgttgcttg gaaaaacaac taccgtaacg ctcactctgc taccacctgg 1080
tctggtcagt acgttggtgg tgctgaagct cgtatcaaca cccagtggct gctgacctct 1140
ggtaccaccg aagctaacgc ttggaaatct accctggttg gtcacgacac cttcaccaaa 1200
gttaaaccgt ctgctgcttc tggtctcgag 1230
Claims (9)
1. The identification method of the canine erythrocyte antigen DEA1.1 is characterized by comprising the following steps: the method comprises the following steps:
step (1), obtaining a dog erythrocyte surface antigen DEA1.1 gene, and performing codon optimization on a DEA1.1 gene sequence by using a gene engineering technology, wherein the sequence is SEQ ID NO. 1;
step (2), a DEA1.1 expression vector is constructed, two enzyme cutting sites are respectively added at two ends of a DEA1.1 gene after codon optimization, the NdeI sites are respectively at the 5 'end, the XhoI sites are at the 3' end, the sequence of the modified DEA1.1 gene is SEQ ID NO.2, and the modified DEA1.1 gene is subjected to gene synthesis and is connected to the corresponding sites of a pET30aCH1-BAB vector;
step (3), transforming the synthesized DEA1.1 gene into DH5 alpha cells for amplification, and extracting amplified plasmids;
step (4), the amplified DEA1.1 gene is transformed into BL21 cells for mass expression, and the supernatant is taken;
step (5), carrying out affinity chromatography purification on DEA1.1 protein, and determining the purity of the protein;
and (6) mixing the DEA1.1 protein obtained after recombinant expression and purification with dog blood, detecting the blood coagulation condition, and identifying the antigenicity of DEA 1.1.
2. The method for identifying canine erythrocyte antigen DEA1.1 according to claim 1, which is characterized in that: the method for transforming the synthesized DEA1.1 gene into DH5 alpha cells for amplification in the step (3) and extracting the amplified plasmid comprises the following steps: DH5 α electroporation competent cells were prepared, and then a vector containing the DEA1.1 gene was transformed into DH5 α electroporation competent cells.
3. The method for identifying canine erythrocyte antigen DEA1.1 according to claim 2, which is characterized in that: the steps for preparing DH5 alpha electrotransformation competent cells are as follows:
diluting the concentration of DH5 alpha strain, and spreading the strain to LB solid culture medium for overnight culture; selecting a single colony grown after overnight culture, and adding the single colony into an LB liquid culture medium for overnight culture; adding overnight cultured bacterial liquid into sterile LB culture medium, culturing until the absorbance of bacterial liquid at OD600 is 0.5-0.8, taking out, cooling in ice water mixture, centrifuging, and collecting bacteria;
washing with pre-cooled ultrapure water and sterile glycerol for several times, discarding the supernatant, resuspending the thallus with glycerol and subpackaging on ice into EP tubes for storage.
4. The method for identifying canine erythrocyte antigen DEA1.1 according to claim 2, which is characterized in that: the steps of transforming the vector containing the DEA1.1 gene into DH5 alpha electrotransfer competent cells are as follows:
adding a vector containing a DEA1.1 gene into a DH5 alpha electrotransformation competent cell, culturing on ice, and transferring the cell into an electroporation container for pulse; then adding LB culture medium into an electroporation container to culture cells, and coating the cells into a screening culture medium for overnight culture; and (4) picking overnight cultured colonies, adding the colonies into an LB screening culture medium for overnight culture, and then extracting amplified plasmids.
5. The method for identifying canine erythrocyte antigen DEA1.1 according to claim 1, which is characterized in that: in the step (4), the amplified DEA1.1 gene is transformed into BL21 cells for mass expression, and the method for collecting the supernatant is as follows: preparing BL21 electrotransformation competent cells, transforming the amplified plasmid into BL21 competent cells, and coating the cells in an LB solid screening culture medium for overnight culture after transformation; taking a single colony for overnight culture, inoculating the single colony into an LB liquid culture medium, culturing until OD600 is 0.5-0.8, and adding IPTG for culture; centrifuging and collecting bacteria; and (3) ultrasonically crushing the thalli, centrifuging at low temperature, taking supernatant, and performing suction filtration by using a micron filter membrane for later use.
6. The method for identifying canine erythrocyte antigen DEA1.1 according to claim 5, which is characterized in that: the BL21 electroporation competent cell preparation steps are as follows:
diluting BL21 strain concentration, spreading to LB solid culture medium for overnight culture; selecting a single colony grown after overnight culture, and adding the single colony into an LB liquid culture medium for overnight culture; adding overnight cultured bacterial liquid into sterile LB culture medium, culturing until the absorbance of bacterial liquid at OD600 is 0.5-0.8, taking out, cooling in ice water mixture, centrifuging, and collecting bacteria;
washing with pre-cooled ultrapure water and sterile glycerol for several times, discarding the supernatant, resuspending the thallus with glycerol and subpackaging on ice into EP tubes for storage.
7. The method for identifying canine erythrocyte antigen DEA1.1 according to claim 5, which is characterized in that: the transformation method for transforming the amplified plasmid into BL21 competent cells is as follows:
adding the amplified DEA1.1 gene into BL21 electrotransformation competent cells, and culturing on ice; transferring to an electroporation container for pulse; adding LB culture medium into an electroporation container to culture cells, and spreading the cells into a screening culture medium for overnight culture; overnight cultured colonies were picked and added to LB selection medium for overnight culture.
8. The method for identifying canine erythrocyte antigen DEA1.1 according to claim 1, which is characterized in that: the method for carrying out affinity chromatography purification on DEA1.1 protein comprises the following steps: and (4) taking the cracked supernatant, passing through a His affinity chromatographic column, and collecting the purified protein.
9. The method for identifying canine erythrocyte antigen DEA1.1 according to claim 1, which is characterized in that: the method for determining the purity comprises the following steps: and (4) carrying out SDS-PAGE electrophoresis on the purified protein, and judging the definition of protein bands according to the purity.
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