CN118406127B - Tropomyosin prokaryotic expression method of Oryza sativa and application thereof - Google Patents
Tropomyosin prokaryotic expression method of Oryza sativa and application thereof Download PDFInfo
- Publication number
- CN118406127B CN118406127B CN202410869793.4A CN202410869793A CN118406127B CN 118406127 B CN118406127 B CN 118406127B CN 202410869793 A CN202410869793 A CN 202410869793A CN 118406127 B CN118406127 B CN 118406127B
- Authority
- CN
- China
- Prior art keywords
- bacterial liquid
- tropomyosin
- prokaryotic expression
- primordial
- supernatant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 102000005937 Tropomyosin Human genes 0.000 title claims abstract description 51
- 108010030743 Tropomyosin Proteins 0.000 title claims abstract description 51
- 230000009465 prokaryotic expression Effects 0.000 title claims abstract description 19
- 238000000034 method Methods 0.000 title claims abstract description 16
- 240000007594 Oryza sativa Species 0.000 title claims description 7
- 235000007164 Oryza sativa Nutrition 0.000 title claims description 7
- 241000244206 Nematoda Species 0.000 claims abstract description 28
- 230000014509 gene expression Effects 0.000 claims abstract description 16
- 239000013612 plasmid Substances 0.000 claims abstract description 15
- 230000006698 induction Effects 0.000 claims abstract description 12
- 229960005486 vaccine Drugs 0.000 claims abstract description 10
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 claims abstract description 7
- 239000007788 liquid Substances 0.000 claims description 54
- 230000001580 bacterial effect Effects 0.000 claims description 53
- 239000006228 supernatant Substances 0.000 claims description 24
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 16
- 108090000623 proteins and genes Proteins 0.000 claims description 13
- 239000008055 phosphate buffer solution Substances 0.000 claims description 12
- 102000004169 proteins and genes Human genes 0.000 claims description 12
- 238000005119 centrifugation Methods 0.000 claims description 10
- 239000000047 product Substances 0.000 claims description 10
- 239000000243 solution Substances 0.000 claims description 9
- 102000003505 Myosin Human genes 0.000 claims description 8
- 108060008487 Myosin Proteins 0.000 claims description 8
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 claims description 7
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 claims description 7
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 claims description 7
- 229930027917 kanamycin Natural products 0.000 claims description 7
- 229960000318 kanamycin Drugs 0.000 claims description 7
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 claims description 7
- 229930182823 kanamycin A Natural products 0.000 claims description 7
- 229960003104 ornithine Drugs 0.000 claims description 7
- 241000894006 Bacteria Species 0.000 claims description 6
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 claims description 6
- 230000009466 transformation Effects 0.000 claims description 6
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims description 5
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims description 5
- 238000001976 enzyme digestion Methods 0.000 claims description 5
- 239000013604 expression vector Substances 0.000 claims description 5
- 239000012634 fragment Substances 0.000 claims description 5
- 239000002244 precipitate Substances 0.000 claims description 5
- 238000012408 PCR amplification Methods 0.000 claims description 4
- 238000001042 affinity chromatography Methods 0.000 claims description 4
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 4
- 150000001413 amino acids Chemical group 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- 102000012410 DNA Ligases Human genes 0.000 claims description 3
- 108010061982 DNA Ligases Proteins 0.000 claims description 3
- 239000012880 LB liquid culture medium Substances 0.000 claims description 3
- 241000209094 Oryza Species 0.000 claims description 3
- 239000012148 binding buffer Substances 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 238000000502 dialysis Methods 0.000 claims description 3
- 239000002609 medium Substances 0.000 claims description 3
- 238000011084 recovery Methods 0.000 claims description 3
- 108091008146 restriction endonucleases Proteins 0.000 claims description 3
- 230000035939 shock Effects 0.000 claims description 3
- 229960000723 ampicillin Drugs 0.000 claims description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 230000001131 transforming effect Effects 0.000 claims description 2
- 239000013598 vector Substances 0.000 claims description 2
- UXOWGYHJODZGMF-QORCZRPOSA-N Aliskiren Chemical compound COCCCOC1=CC(C[C@@H](C[C@H](N)[C@@H](O)C[C@@H](C(C)C)C(=O)NCC(C)(C)C(N)=O)C(C)C)=CC=C1OC UXOWGYHJODZGMF-QORCZRPOSA-N 0.000 claims 2
- 229960004601 aliskiren Drugs 0.000 claims 2
- 241000233866 Fungi Species 0.000 claims 1
- 238000010367 cloning Methods 0.000 claims 1
- 239000002773 nucleotide Substances 0.000 abstract description 12
- 125000003729 nucleotide group Chemical group 0.000 abstract description 12
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 abstract description 4
- 238000005215 recombination Methods 0.000 abstract description 4
- 230000006798 recombination Effects 0.000 abstract description 4
- 238000000338 in vitro Methods 0.000 abstract description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 30
- 230000001939 inductive effect Effects 0.000 description 15
- 241001494479 Pecora Species 0.000 description 14
- 238000012360 testing method Methods 0.000 description 12
- 108020004705 Codon Proteins 0.000 description 10
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 10
- 238000005457 optimization Methods 0.000 description 8
- 241000243795 Ostertagia Species 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 235000013601 eggs Nutrition 0.000 description 5
- 238000010828 elution Methods 0.000 description 5
- 210000003608 fece Anatomy 0.000 description 5
- 230000003053 immunization Effects 0.000 description 5
- 238000002649 immunization Methods 0.000 description 5
- 238000001556 precipitation Methods 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 238000003776 cleavage reaction Methods 0.000 description 4
- 230000007017 scission Effects 0.000 description 4
- 241000243770 Bursaphelenchus Species 0.000 description 3
- 241000238631 Hexapoda Species 0.000 description 3
- 240000002853 Nelumbo nucifera Species 0.000 description 3
- 235000006508 Nelumbo nucifera Nutrition 0.000 description 3
- 235000006510 Nelumbo pentapetala Nutrition 0.000 description 3
- 208000030852 Parasitic disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 244000144972 livestock Species 0.000 description 3
- 244000045947 parasite Species 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 241000499865 Ornithogalum Species 0.000 description 2
- 240000005859 Orthosiphon aristatus Species 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 239000003096 antiparasitic agent Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 230000017188 evasion or tolerance of host immune response Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 239000000273 veterinary drug Substances 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 241001466077 Salina Species 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000011217 control strategy Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 210000001198 duodenum Anatomy 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 230000002550 fecal effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 239000010871 livestock manure Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000004118 muscle contraction Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- 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
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
- C07K14/4354—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0003—Invertebrate antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
- A61P33/10—Anthelmintics
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/70—Vectors or expression systems specially adapted for E. coli
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Veterinary Medicine (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Public Health (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a prokaryotic expression method of tropomyosin of an Olympic cervical nematode and application thereof, wherein the prokaryotic expression method is characterized in that the nucleotide sequence of the tropomyosin of the Olympic cervical nematode is artificially synthesized, is connected with a pET-28a carrier, two restriction sites of NcoI and XhoI and 6 histidine tags are added, a recombinant plasmid is constructed, and the recombinant plasmid is transformed into escherichia coli BL21 (DE 3) and is subjected to induction recombination for high-efficiency expression, so that the recombinant tropomyosin of the Olympic cervical nematode in vitro is obtained, and is used for preparing an Olympic cervical nematode vaccine.
Description
Technical Field
The invention belongs to the technical field of biological control, and particularly relates to a tropomyosin prokaryotic expression method of an Olympic cervical nematode and application thereof.
Background
In recent years, the risk of parasitic diseases has become increasingly prominent, and these disorders are often overlooked by humans because of the less pronounced clinical manifestations. Development of antiparasitic drugs is currently slow, but the accumulation of resistance of nematodes is very rapid, and the antiparasitic drugs available in veterinary clinics are very limited, so that the control strategy of parasitic diseases must be developed toward more sustainable direction. The vaccine as a preventive measure has obvious advantages in the aspects of disease occurrence reduction, long-term protection, no veterinary drug residue and the like. However, many challenges are faced in the development of parasite vaccines, such as the problems of multicellular organisms, complex life cycle, ingenious immune escape mechanism, and large heterogeneity of host population immune response, so that the development and application of the vaccine are relatively behind those of infectious diseases.
In the early research, tropomyosin (TM) of the Ornithine (Nemafodirus oiratianus Rajewskaja) has obvious differential expression (more than 1430 times of average differential expression) in each larva and adult development stage compared with the egg group, and shows the potential value of the Tropomyosin as vaccine antigen. As a key protein in the muscle tissue of a variety of parasites, TM is involved not only in muscle contraction, but also possibly in parasite movement, tissue invasion and immune escape. Therefore, the invention prevents and treats the digestive tract nematode disease by developing the vaccine based on TM, is hopeful to reduce the infection rate of the digestive tract nematode of livestock, reduce the dependence on medicines and the occurrence of drug resistance, reduce veterinary drug residues and environmental pollution, also provides important basic research data for sustainable development of livestock industry in China, effectively prevents and controls the parasitic disease of livestock, accelerates the peasants to get rid of poverty and become rich.
Disclosure of Invention
The first object of the present invention is to provide tropomyosin of an Olympic cervical nematode.
A second object of the present invention is to provide a method for prokaryotic expression of tropomyosin in Ostertagia sp.
A third object of the present invention is to provide the use of tropomyosin of the species of the cervical nematode of australian.
The first object of the invention is implemented by the following technical scheme:
An Olympic cervical nematode tropomyosin has an amino acid sequence shown in SEQ ID NO: 1.
Further, the primordial myosin of the Oryza sativa is obtained by inserting a nucleotide sequence of the primordial myosin as a target protein into a pET-28a prokaryotic expression vector, constructing, inducing expression recombination and purifying; the nucleotide sequence of the tropomyosin is a nucleotide sequence subjected to codon optimization, and the nucleotide sequence before codon optimization is shown as SEQ ID NO:2, the nucleotide sequence after codon optimization is shown as SEQ ID NO:3 is shown in the figure; the codon optimization includes insertion of the NcoI cleavage site CCATGG, xhoI cleavage site CTCGAG, 6 histidine sites and stop codon TGA.
The second object of the invention is implemented by the following technical scheme:
a method for prokaryotic expression of the tropomyosin of the cervical nematode of Olympic, comprising the steps of:
S1, the sequence represented by SEQ ID NO:2 as a template, carrying out PCR amplification by using a designed primer to obtain a PCR product fragment;
S2, constructing an expression vector; the step S2 comprises the following steps: s21, respectively carrying out double enzyme digestion on the PCR product fragment obtained in the S1 and pET-28a plasmid by using restriction enzymes NcoI and XhoI, and carrying out agarose gel electrophoresis recovery; s22, connecting the double enzyme digestion products obtained in the S21 by using T4 DNA ligase to obtain recombinant plasmids; s23, transforming the recombinant plasmid into competent cells of escherichia coli BL21 (DE 3), and culturing to obtain competent bacterial liquid; s24, carrying out transformation culture on the competent bacterial liquid to obtain a transformed bacterial liquid;
s3, IPTG induction expression recombinant fusion protein; the step S3 comprises the following steps: s31, performing induced expression on the transformed bacterial liquid to obtain an induced bacterial liquid; s32, carrying out ultrasonic crushing and centrifugation on the induced bacterial liquid, the resuspension;
S4, purifying the supernatant after centrifugation in the S32; the step S4 comprises the following steps: s41, passing the supernatant through a Ni-NTA affinity chromatographic column, eluting target proteins, and collecting effluent; s42, adding the effluent into a dialysis bag, and dialyzing by using PBS (phosphate buffer solution) for overnight to obtain the primordial myosin of the Oryza parviflora.
Further, the step S23 is specifically that 1. Mu.L of plasmid is added into 100. Mu.L of competent bacteria of escherichia coli BL21 (DE 3) and placed on ice with a temperature of 20 min; heat shock 90 sec at 42 ℃ and then rapidly placed in ice 5 min; then adding the strain into LB culture solution, and shaking for 1 h at 37 ℃ and 220 r/min to obtain competent bacteria solution;
Further, the step S24 is specifically that the competent bacterial liquid obtained in the step S23 is centrifuged and then is coated on a conversion plate, wherein the conversion plate is an LB plate culture medium with ampicillin resistance or kanamycin resistance, and is subjected to inversion culture at 37 ℃ for overnight; the monoclonal on the transformation plate is picked up, inoculated in LB liquid medium with kanamycin resistance, shaken overnight at 37 ℃ under 220 r/min, and the following day according to volume 1:100 is inoculated in a LB liquid culture medium with kanamycin resistance, and is shaken under the conditions of 37 ℃ and 220 r/min until the OD 600 of the transformed bacterial liquid is 0.5-0.8.
Further, the step S31 is specifically that IPTG induction 18 h is added into the transformed bacterial liquid obtained in the step S24; IPTG concentration is 0.2-0.5 mM, and induction temperature is 16-30 ℃.
Further, the step S32 is specifically that the induced bacterial liquid obtained in the step S31 is centrifuged for 10 min at 4000 r/min and room temperature, the supernatant is discarded, PBS is added according to 1/10 of the volume of the induced bacterial liquid to resuspend bacterial precipitation, PMSF with the final concentration of 1mM is added into the resuspension, and then ultrasonic crushing and centrifugation are carried out.
Further, the step S41 is specifically that the Ni-NTA affinity chromatography column is balanced by Ni-NTA Binding-Buffer, and then the supernatant is loaded to the Ni-NTA affinity chromatography column at a flow rate of 1 mL/min; the target protein was eluted with Ni-NTA filtration-Buffer at a flow rate of 1 mL/min, and the effluent was collected.
The third object of the present invention is implemented by the following technical scheme:
the application of the primordial myosin of the Oryza sativa in preparing the vaccine of the Oryza sativa.
The beneficial effects are that: the invention provides a prokaryotic expression method of tropomyosin of an Olympic cervical nematode, which is characterized in that a nucleotide sequence of the tropomyosin of the Olympic cervical nematode is artificially synthesized, the tropomyosin is connected with a pET-28a vector, two restriction sites of NcoI and XhoI and 6 histidine tags are added, a recombinant plasmid is constructed, the recombinant plasmid is transformed into escherichia coli BL21 (DE 3), and the recombinant tropomyosin in vitro of the Olympic cervical nematode is obtained through induction recombination and high-efficiency expression, and the recombinant tropomyosin can be used for preparing an Olympic cervical nematode vaccine.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a SDS-PAGE map of the expression products of different induction conditions in example 2; in the figure: m, protein markers; 1. inducing a bacterial liquid A supernatant; 2. inducing bacterial liquid A precipitation liquid; 3. inducing a bacterial liquid B supernatant; 4. inducing bacterial liquid B precipitation liquid; 5. inducing bacterial liquid C supernatant; 6. inducing bacterial liquid C precipitation liquid; 7. inducing bacterial liquid D supernatant; 8. and (5) inducing a bacterial solution D precipitation solution.
FIG. 2 is a SDS-PAGE chart of the supernatant purified products of example 2 at different stages; in the figure: m, SDS-PAGE MARKER; 1. supernatant; 2 and 3, passing through Ni-NTA affinity chromatographic column; 4. 25mM imidazole elution; 5. after elution with 50mM imidazole; 6. 100mM imidazole elution; 7. after 500mM imidazole elution.
FIG. 3 is a SDS-PAGE of the tropomyosin of the plant of the Ostertagia sp.3 species; in the figure: m, SDS-PAGE MARKER; 1. tropomyosin.
FIG. 4 is a Western Blot diagram of the tropomyosin of Ostertagia ensiformis in example 4; in the figure: m, protein markers; 1. primordial myosin of the Oryza parvula; 2. negative control.
Fig. 5 shows the daily gain of sheep in the test and control groups of example 4.
FIG. 6 is a graph showing the number of eggs per gram of fecal worm in the sheep of the test and control groups of example 4.
Figure 7 shows sheep manure dry matter content for the test and control groups of example 4.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The experiments in the embodiment of the invention are all set with three repetitions, and the results are averaged.
Example 1: method for prokaryotic expression of the Ostertagia pinnata TM.
A prokaryotic expression method of an Olympic cervical nematode TM comprises the steps S1-S4.
S1, SEQ ID NO:2 as a template, and carrying out PCR amplification by using designed primers (the primer sequences are shown in SEQ ID NO: 4-SEQ ID NO: 21) to obtain a PCR product fragment.
S2, constructing an expression vector; s2 comprises the following steps: s21, respectively double-enzyme cutting the PCR product fragment obtained in the S1 and pET-28a plasmid by using restriction enzymes NcoI and XhoI, and carrying out agarose gel electrophoresis and recovery; s22, connecting the double enzyme digestion products obtained in the S21 by using T4 DNA ligase to obtain recombinant plasmids; s23, adding 1 mu L of plasmid into 100 mu L of competent bacteria of escherichia coli BL21 (DE 3), and placing the mixture on ice for 20 min; heat shock 90 sec at 42 ℃ and then rapidly placed in ice 5 min; then adding 600 mu L of LB culture solution, and shaking at 37 ℃ and 220 r/min for 1 to h to obtain competent bacteria solution; s24, centrifuging the competent bacterial liquid obtained in the S23, coating all the competent bacterial liquid on a conversion plate, wherein the conversion plate is an LB plate culture medium containing 50 mug/mL Amp or Kan, and culturing the culture medium upside down at 37 ℃ for overnight; selecting a monoclonal on a conversion plate, inoculating the monoclonal on a LB liquid medium containing 50 mug/mL Kan of 4 mL, shaking overnight at 37 ℃ and 220 r/min, and carrying out volume 1 the following day: 100 is inoculated in LB liquid culture medium with kanamycin resistance, and is shaken under the conditions of 37 ℃ and 220 r/min until the OD 600 of the transformed bacterial liquid is 0.5-0.8.
S3, IPTG induction expression recombinant fusion protein; s3 comprises the following steps: s31, adding IPTG to the transformed bacterial liquid obtained in the step S24 to induce 18 h; IPTG concentration is 0.2-0.5 mM, induction temperature is 16-30 ℃, and induced bacterial liquid is obtained; s32, centrifuging the induced bacterial liquid obtained in the step S31 at 4000r/min and room temperature for 10: 10 min, discarding the supernatant, adding PBS according to 1/10 of the volume of the induced bacterial liquid to resuspend bacterial precipitate, adding PMSF with the final concentration of 1 mM into the resuspension, and then carrying out ultrasonic crushing and centrifugation.
S4, purifying the supernatant after centrifugation in S32; s4 comprises the following steps: s41, balancing the Ni-NTA affinity chromatographic column by using Ni-NTA Binding-Buffer (PBS, pH 7.4), and loading the supernatant to the Ni-NTA affinity chromatographic column at a flow rate of 1 mL/min; eluting target protein with Ni-NTA solution-Buffer (PBS, 500 mM imidazole, pH 7.4) at flow rate of 1 mL/min, and collecting effluent; s42, adding the effluent into a dialysis bag, dialyzing by using PBS (phosphate buffer solution) for overnight to obtain the Olicomia salina TM, wherein the amino acid sequence of the TM is shown as SEQ ID NO: 1.
Example 2: SDS-PAGE analysis.
(1) Expression identification of IPTG induced recombinant bacterium fusion protein.
Taking samples with OD 600 of 0.5-0.6 from the transformed bacterial liquid obtained in the S24 in the embodiment 1, and respectively inducing fusion protein expression in A, B, C, D groups, wherein the following specific steps are as follows: A. adding IPTG into the transformed bacterial liquid, and inducing for 18 h at 16 ℃ with the concentration of 0.2 mM to obtain an induced bacterial liquid A; B. adding IPTG into the transformed bacterial liquid, and inducing for 18 h at 20 ℃ with the concentration of 0.2 mM to obtain an induced bacterial liquid B; C. adding IPTG into the transformed bacterial liquid, and inducing for 18 h at 30 ℃ with the concentration of 0.2 mM to obtain an induced bacterial liquid C; D. adding IPTG into the transformed bacterial liquid, and inducing for 18 h at 20 ℃ to obtain the induced bacterial liquid D, wherein the concentration of the IPTG is 0.5 mM.
Taking 1mL of induced bacterial liquid A, B, C, D, centrifuging at 10000 r/min and room temperature for 2 min, discarding supernatant, re-suspending bacterial precipitate re-suspension by 100 mu L of PBS, adding PMSE with the final concentration of 1mM into the re-suspension, performing ultrasonic crushing, centrifuging, taking supernatant and precipitate, adding TBS into the supernatant and the precipitate respectively for re-suspension, performing 12% SDS-PAGE, and detecting and analyzing Coomassie brilliant blue staining and developing bands.
As shown in FIG. 1, the supernatant was expressed more than the pellet, and it was seen that the recombinant protein was soluble expressed in E.coli; in addition, the expression of the induced bacterial liquid B was more than that of the induced bacterial liquid A, C, D, which indicates that the induction temperature of 20 ℃ was 18: 18 h and the IPTG concentration was 0.2: 0.2 mM, which are more suitable conditions for the induction expression.
(2) Purification of recombinant fusion proteins.
SDS-PAGE analysis was performed on the supernatant after centrifugation in S32 in example 1, and the effluent after passing the supernatant in S41 through a Ni-NTA affinity column (in two parallel), after eluting with 25mM imidazole, after eluting with 50mM imidazole, after eluting with 100mM imidazole, and after eluting with 500mM imidazole, as shown in FIG. 2, the supernatant after centrifugation in S32 was most expressed, but the impurity protein was more before purification by the Ni-NTA affinity column; after 500mM imidazole elution, the protein was expressed more and the purity was highest.
Example 3: orichthyophthiriasis TM.
An Olicomia cervical nematode TM with an amino acid sequence shown in SEQ ID NO: 1.
The invention discloses an Olympic cervical nematode TM which is obtained by inserting a nucleotide sequence of tropomyosin as a target protein into a pET-28a prokaryotic expression vector, constructing, inducing expression recombination and purifying; the SDS-PAGE electrophoresis diagram is shown in figure 3, and the in vitro prokaryotic expression purity of TM (4 mug) of the Ornithine is more than 85 percent by the judgment of SDS-PAGE, and the construction is successful. The nucleotide sequence of tropomyosin is a nucleotide sequence subjected to codon optimization, and the nucleotide sequence before codon optimization is shown as SEQ ID NO:2 (which is obtained from the inventor collecting the Ornithogalum estuary and the Ornithogalum grandiflorum Nemafodirus oiratianus Rajewskaja of the Erdos municipality, the nucleotide sequence after codon optimization is shown as SEQ ID NO:3 is shown in the figure; codon optimization includes insertion of the NcoI cleavage site CCATGG, xhoI cleavage site CTCGAG, 6 histidine sites and stop codon TGA.
Example 4: application of Oryza glutinosa TM is provided.
(1) Western bolt analysis.
The Olympic cervical nematode TM of example 3 was used as antigen, sheep serum naturally infected with Olympic cervical nematode was used as primary antibody, and rabbit anti-sheep IgG-HRP was used as secondary antibody.
As shown in the WB results of FIG. 4, a specific band appeared between the relative molecular weights 38-52 KDa, whereas no band was detected by the negative control, indicating that recombinant TM protein (6. Mu.g), namely, the Olympic Bursaphelenchus of the present invention, was recognized by sheep serum naturally infected with Olympic Bursaphelenchus.
(2) Immune protective test against sheep.
10 Healthy sheep were randomly divided into 2 groups, 5 sheep each in the test group and the control group, and immunized as shown in Table 1. After the first immunization is mixed and emulsified by using the Olympic cervical nematode TM antigen and the Freund's complete adjuvant, the inguinal subcutaneous multipoint injection is carried out, and after 14 d, the second immunization is carried out by using the Freund's incomplete adjuvant and the Olympic cervical nematode TM antigen, the back subcutaneous multipoint injection is carried out; after the second immunization of 14 d, 10000 infectious larvae are infected by the Ornithine, and the mouthfeel is changed.
TABLE 1 Experimental grouping and immunization Condition
The weight daily gain, the number of eggs per gram of faeces, the dry matter content of faeces and the like of sheep in the test group and the control group are counted, and the results are shown in figures 5-7, and the corresponding data are the average value of the data of 5 sheep in each group. It can be seen that immunization of the Ornithine (TM) had no effect on daily gain of sheep (P > 0.05), and that daily gain of sheep in the test group was significantly higher than that in the control group (P < 0.05) after challenge; the number of faecal eggs can be reduced after the Olympic cervical nematode TM is immunized, and the average egg reduction rate is 85.7 percent (P is less than 0.05); the test group immunized with the Olympic Bursaphelenchus has relatively stable dry matter content of the feces of the test group, and the control group has relatively low dry matter content of the feces and a certain diarrhea tendency.
After infection with 28 d of the Olympic cervical nematodes, 3 sheep were randomly selected from each of the test group and the control group, the duodenum was dissected, and adult counts were collected, and the results are shown in Table 2.
TABLE 2 adult counting results
As shown in Table 2, the average number of the lotus insects in the control group is 352, the average number of the lotus insects in the test group immunized with the Ostertagia virginiana TM is 78, the average reduction rate of the lotus insects is 77.8%, and the t-test analysis shows that the difference between the test group and the control group is remarkable (P < 0.05).
The experiment proves that the Orthosiphon aristatus TM has certain resistance to sheep infected with the Orthosiphon aristatus.
In conclusion, the Olympic cervical nematode TM can be applied to the preparation of Olympic cervical nematode vaccine.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (8)
1. An Olympic cervical nematode tropomyosin, which has an amino acid sequence as shown in SEQ ID NO: 1.
2. The method for prokaryotic expression of the tropomyosin of the Ornithina parvulus of claim 1, comprising the steps of:
S1, the sequence represented by SEQ ID NO:2 as a template, performing PCR amplification by using the designed primer, and amplifying the sequence of SEQ ID NO:2 and cloning the gene sequence into a vector pcoldII, and carrying out agarose gel electrophoresis on a PCR product obtained by PCR amplification, and recovering and purifying;
S2, constructing an expression vector; the step S2 comprises the following steps: s21, respectively carrying out double enzyme digestion on the PCR product fragment obtained in the S1 and pET-28a plasmid by using restriction enzymes NcoI and XhoI, and carrying out agarose gel electrophoresis recovery; s22, connecting the double enzyme digestion products obtained in the S21 by using T4 DNA ligase to obtain recombinant plasmids; s23, transforming the recombinant plasmid into competent cells of escherichia coli BL21 (DE 3), and culturing to obtain competent bacterial liquid; s24, carrying out transformation culture on the competent bacterial liquid to obtain a transformed bacterial liquid;
s3, IPTG induction expression recombinant fusion protein; the step S3 comprises the following steps: s31, performing induced expression on the transformed bacterial liquid to obtain an induced bacterial liquid; s32, carrying out ultrasonic crushing and centrifugation on the induced bacterial liquid, the resuspension;
S4, purifying the supernatant after centrifugation in the S32; the step S4 comprises the following steps: s41, passing the supernatant through a Ni-NTA affinity chromatographic column, eluting target proteins, and collecting effluent; s42, adding the effluent into a dialysis bag, and dialyzing by using PBS (phosphate buffer solution) for overnight to obtain the primordial myosin of the Oryza parviflora.
3. The prokaryotic expression method of the primordial tropomyosin from the Oryza sativa according to claim 2, wherein S23 is characterized by adding 1 μl of plasmid into 100 μl of competent bacteria of Escherichia coli BL21 (DE 3) and placing on ice 20 min; heat shock 90 sec at 42 ℃ and then rapidly placed in ice 5 min; then adding the strain into LB culture solution, and shaking at 37 ℃ and 220 r/min for 1 to h to obtain the competent fungus solution.
4. The prokaryotic expression method of the primordial myelegans tropomyosin according to claim 2, wherein the step S24 is that the competent bacterial liquid obtained in the step S23 is centrifuged and then coated on a transformation plate, and the transformation plate is an ampicillin-resistant or kanamycin-resistant LB plate culture medium, and is cultured upside down at 37 ℃ overnight; the monoclonal on the transformation plate is picked up, inoculated in LB liquid medium with kanamycin resistance, shaken overnight at 37 ℃ under 220 r/min, and the following day according to volume 1:100 is inoculated in a LB liquid culture medium with kanamycin resistance, and is shaken under the conditions of 37 ℃ and 220 r/min until the OD 600 of the transformed bacterial liquid is 0.5-0.8.
5. The prokaryotic expression method of the primordial myosin of the Ornithine according to claim 2, wherein the step S31 is specifically that IPTG is added to the transformed bacterial liquid obtained in the step S24 to induce 18 h; IPTG concentration is 0.2-0.5 mM, and induction temperature is 16-30 ℃.
6. The method for prokaryotic expression of the tropomyosin from the Ornithine according to claim 2, wherein the step S32 is specifically that the induced bacterial liquid obtained in the step S31 is centrifuged at 4000 r/min at room temperature for 10 min, the supernatant is discarded, PBS is added according to 1/10 of the volume of the induced bacterial liquid to resuspend bacterial precipitate, PMSF with a final concentration of 1 mM is added to the resuspension, and then ultrasonic disruption and centrifugation are performed.
7. The prokaryotic expression method of the primordial tropomyosin of the Ornithine according to claim 2, wherein the S41 is specifically that the Ni-NTA affinity chromatography column is equilibrated with Ni-NTA Binding-Buffer, and then the supernatant is loaded onto the Ni-NTA affinity chromatography column at a flow rate of 1 mL/min; the target protein was eluted with Ni-NTA filtration-Buffer at a flow rate of 1 mL/min, and the effluent was collected.
8. Use of the primordial myosin of an aliskiren according to claim 1 in the preparation of an aliskiren vaccine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410869793.4A CN118406127B (en) | 2024-07-01 | 2024-07-01 | Tropomyosin prokaryotic expression method of Oryza sativa and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410869793.4A CN118406127B (en) | 2024-07-01 | 2024-07-01 | Tropomyosin prokaryotic expression method of Oryza sativa and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN118406127A CN118406127A (en) | 2024-07-30 |
| CN118406127B true CN118406127B (en) | 2024-09-10 |
Family
ID=92001422
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410869793.4A Active CN118406127B (en) | 2024-07-01 | 2024-07-01 | Tropomyosin prokaryotic expression method of Oryza sativa and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118406127B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101508731A (en) * | 2008-12-31 | 2009-08-19 | 吉林大学 | Trichinella spiralis and tumor-associated antigen protein, preparation and medical use thereof |
| AU2020102009A4 (en) * | 2020-08-27 | 2020-10-01 | Nanjing Agricultural University | In vitro expression of pear PbrRALF2 protein and preparation method of polyclonal antibody thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636149B (en) * | 2017-01-03 | 2019-10-25 | 内蒙古农业大学 | Parabronema skrjabini serine/threonine protein kitase Pj_STPK gene cloning, prokaryotic expression method and application |
| CN107312786B (en) * | 2017-07-14 | 2019-10-29 | 内蒙古农业大学 | A kind of preparation method and application recombinating Parabronema skrjabini cysteine proteinase |
-
2024
- 2024-07-01 CN CN202410869793.4A patent/CN118406127B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101508731A (en) * | 2008-12-31 | 2009-08-19 | 吉林大学 | Trichinella spiralis and tumor-associated antigen protein, preparation and medical use thereof |
| AU2020102009A4 (en) * | 2020-08-27 | 2020-10-01 | Nanjing Agricultural University | In vitro expression of pear PbrRALF2 protein and preparation method of polyclonal antibody thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN118406127A (en) | 2024-07-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP2002511492A (en) | Proboscis | |
| CN102276730B (en) | Preparation method for staphylococcus aureus Iron-regulated surface determinant B immunodominant fragment (IsdBid)-target of RNAIII activating protein (TRAP) fusion protein and application thereof | |
| CN113637056A (en) | Kit for identifying Brucella melitensis and Brucella melitensis of other species | |
| CN107759674B (en) | Mycoplasma bovis immunity-related protein, detection kit containing protein and application of protein in mycoplasma bovis antibody detection | |
| CN110845582B (en) | Preparation of feline parvovirus recombinant protein and monoclonal antibody thereof | |
| CN109486846B (en) | Three-gene recombinant plasmid of Brucella, construction method and expression and application thereof in escherichia coli | |
| CN109777785B (en) | Hybridoma cell strain and application thereof | |
| CN118406127B (en) | Tropomyosin prokaryotic expression method of Oryza sativa and application thereof | |
| CN113481170A (en) | Hybridoma cell strain, antibody produced by hybridoma cell strain and preparation method of antibody | |
| CN114288402B (en) | Preparation method and application of mycoplasma hyopneumoniae multi-epitope genetic engineering subunit vaccine based on reverse vaccinology technology | |
| CN113278069B (en) | Polyclonal antibody of Chlamydomonas reinhardtii intraciliary transport protein IFT38, application and method | |
| CN111961121B (en) | Clostridium perfringens epsilon toxin mutant protein, preparation method, application and vaccine thereof | |
| CN111978380A (en) | Wild type clostridium emphysema cytotoxin A as well as preparation method, application and vaccine thereof | |
| CN111398604A (en) | Serological detection method of Buffalo A19-delta VirB12 vaccine | |
| CN104402974A (en) | Polypeptide with mucosal immunity adjuvant activity, and application of polypeptide in preparation of mucosal immunity adjuvant | |
| CN116574160B (en) | Streptococcus suis antigen protein and application thereof | |
| CN110117607A (en) | A kind of recombinant vector and expression of ustilago zeae effect protein Pit2 gene | |
| CN116041541B (en) | Mycobacterium tuberculosis antigen EPPA011 and application thereof | |
| CN110423770A (en) | A kind of recombinant vector and expression of ustilago zeae effect protein CMU1 gene | |
| CN117003888B (en) | Enterotoxin-producing escherichia coli antigen multi-epitope fusion protein and preparation method and application thereof | |
| CN115850523B (en) | Herpes simplex virus I-type specific fusion protein antigen, preparation method thereof and detection kit | |
| CN116640755B (en) | Streptococcus prophage lyase lys1519 and application thereof | |
| CN116813767A (en) | FGF21 protein polyclonal antibody preparation method | |
| CN103087189A (en) | Method for obtaining polyclonal antibody of sheep independent growth factor 1B | |
| CN117004672A (en) | Method for preparing homogeneous multimeric protein by prokaryotic expression system |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |