CN117003898B - LHRH4-CRM197-LF multimeric recombinant protein and preparation method and application thereof - Google Patents
LHRH4-CRM197-LF multimeric recombinant protein and preparation method and application thereof Download PDFInfo
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- CN117003898B CN117003898B CN202311253686.0A CN202311253686A CN117003898B CN 117003898 B CN117003898 B CN 117003898B CN 202311253686 A CN202311253686 A CN 202311253686A CN 117003898 B CN117003898 B CN 117003898B
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- lhrh4
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Classifications
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- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/23—Luteinising hormone-releasing hormone [LHRH]; Related peptides
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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
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
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- 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
- C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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Abstract
The invention discloses LHRH4-CRM197-LF multimeric recombinant protein and a preparation method and application thereof, belonging to the technical field of bioengineering; the multimeric recombinant protein structure comprises a LHRH4 polymer, a flexible peptide 1, a recombinant CRM197 protein, a flexible peptide 2 and a recombinant lactoferrin which are connected in series, has a composite structure composed of various proteins, can effectively induce the production of LHRH antibodies by orally or injecting immune mammals, can generate more durable and obvious function of inhibiting reproductive capacity, and has important functions on reproductive regulation, hormone regulation, medical research and clinical application of the mammals.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to LHRH4-CRM197-LF multimeric recombinant protein, and a preparation method and application thereof.
Background
Luteinizing hormone releasing hormone (Luteinizing Hormone-Releasing Hormone, LHRH) is a hormone secreted by the hypothalamus. LHRH polypeptide enters anterior pituitary through hypothalamus-pituitary-portal vein system, combines with pituitary gonadotrophin secreting cell specific receptor to generate gonadotrophin (GtH), stimulates release of GtH, regulates secretion of Follicle Stimulating Hormone (FSH), luteinizing hormone (Luteinizing Hormone, LH), stimulates female gonad (ovary) development, follicle maturation and ovulation, and also promotes male gonad (testis) development, testosterone secretion and spermatogenesis, thereby playing a role in regulating vertebrate reproduction.
Along with the development of gene recombination technology and Hormone immunology, hormone immune neutralization technology (Hormone ImmuNOneutralization, HIN) is becoming an important tool for endocrine research, and the concept of LHRH vaccine is also generated, and the basic principle is as follows: the exogenous LHRH is used for actively immunizing the mammal, and the immune system is induced to generate excessive LHRH specific antibodies which are combined with endogenous LHRH so as to ensure that the endogenous LHRH loses biological activity, interfere hypothalamus-pituitary gland-gonadal axis function and inhibit release of LH and FSH, thereby playing a role in regulating reproductive activity of the mammal.
Since mammalian LHRH is a short peptide of 10 amino acids, it needs to be conjugated as a hapten to a foreign carrier protein to effectively stimulate the immune system to produce LHRH antibodies. In the early stages of LHRH immunotherapy, LHRH standards or LHRH analogues may be coupled to carrier proteins by carbodiimide methods, glutaraldehyde methods, m-maleimidobozyl-N-hydroxysuccinimide (MBS) methods. For example, the carbodiimide method can be used to couple LHRH analogue monomers with human albumin (Human Serum Albumin, HAS) and to immunize bulls using freund's adjuvant. Glutaraldehyde method can be used to couple LHRH analog monomers with PAP protein (pokeweed antiviral protein, PAP) to immunize puppies. Because the LHRH monomer has the same structure as the endogenous LHRH molecule, the effect of immunization of mammals after chemical coupling of the LHRH monomer and carrier protein is not stable. And the lack of biological activity of the LHRH monomer expressed by the prokaryote has no application value. At present, it is reported that LHRH gene sequence and hepatitis B virus (Hepatitis B virus) surface antigen (HBsAg) gene are inserted into silkworm polyhedrin virus vector through genetic engineering, recombinant virus insect cells and silkworm are used for expressing HBsAg-LHRH recombinant protein, but the expression level of the recombinant protein expressed by virus is low, and the recombinant protein cannot be put into application.
In the prior art, the durability and the significance of the recombinant protein related to LHRH on the fertility regulation are not high enough, the in vivo stability is not high, and the generation of LHRH antibodies is induced by injection mostly, so that no report related to oral administration is seen.
Disclosure of Invention
The invention aims to solve the technical problems that the recombinant protein of LHRH in the prior art is not high enough in durability and significance for regulating and controlling reproductive capacity and has poor effect for regulating and controlling the level of LHRH antibodies, and provides the LHRH4-CRM197-LF multimeric recombinant protein, a preparation method and application thereof.
The invention is realized by the following technical scheme.
The invention provides an LHRH4-CRM197-LF multimeric recombinant protein, the structure of which comprises an LHRH4 polymer, a flexible peptide 1, a recombinant CRM197 protein, a flexible peptide 2 and a recombinant lactoferrin which are connected in series.
The invention designs a novel expression vector system pGEX-LHRH4-linker1-CRM197-linker2-LF for simultaneously expressing LHRH4 polymer, recombinant diphtheria toxin nontoxic mutant CRM197 protein, recombinant lactoferrin and flexible peptide, and the LHRH4-CRM197-LF multimeric recombinant protein is obtained through expression of the expression vector, and has uniqueness and specificity in structure.
Under the coordination mechanism of LHRH4 polymer, recombinant diphtheria toxin nontoxic mutant CRM197 protein and recombinant lactoferrin, the multimeric recombinant protein can effectively induce the production of LHRH antibodies by orally taking or injecting the mammal, can generate more durable and obvious function of inhibiting reproductive capacity, and has important functions on reproductive regulation, hormone regulation, medical research and clinical application of the mammal.
As a further technical scheme, the gene for encoding the LHRH4-CRM197-LF multimeric recombinant protein has a nucleotide sequence shown as SEQ ID NO. 1.
As a further technical scheme, the LHRH4-CRM197-LF multimeric recombinant protein has an amino acid sequence shown as SEQ ID NO. 2.
The invention also provides a preparation method of the LHRH4-CRM197-LF multimeric recombinant protein, which comprises the following steps:
s1, designing and obtaining a nucleotide sequence shown as SEQ ID NO. 1;
s2, constructing recombinant plasmids: connecting a nucleotide sequence shown in SEQ ID NO.1 with a vector subjected to double enzyme digestion, converting the connected product into E.coli DH5 alpha competent cells, and performing amplification culture on the E.coli DH5 alpha to extract recombinant plasmids;
s3, expression and purification of a multimeric recombinant protein: the recombinant plasmid is transformed into genetic engineering bacteria, the expression of the recombinant protein is induced by IPTG, and the recombinant protein is obtained through separation and purification.
As a further technical scheme, the construction of the recombinant plasmid in step S2 includes:
s21, extracting pGEX-5X-1-H plasmid;
s22, performing double enzyme digestion by using a double enzyme digestion system BamHI enzyme and EcoRI enzyme to obtain digested pGEX-5X-1-H;
s23, connecting the digested pGEX-5X-1-H with a nucleotide sequence shown in SEQ ID NO.2 under the action of T4 DNA ligase to obtain a connection product;
s24, after the connection product is transformed into E.coli DH5 alpha competent cells, E.coli DH5 alpha is subjected to expansion culture, and recombinant plasmids are extracted.
As a further technical solution, step S22 includes:
use carrier double enzyme system BamHI enzyme 0.5 mu L, ecoRI enzyme 0.5 mu L, pGEX-5X-1-H carrier 6 mu g, 10xBuffer R2 mu L, ddH 2 O10 mu L, carrying out constant temperature double enzyme digestion for 2H at 37 ℃, carrying out agarose gel electrophoresis after double enzyme digestion, and purifying pGEX-5X-1-H after enzyme digestion.
As a further technical scheme, the step S3 of expressing and purifying the recombinant multimeric protein comprises:
s31, transforming the recombinant plasmid into E.coli BL21 gene engineering bacteria, culturing the bacteria to a logarithmic growth phase in an oscillating way, and treating the bacteria by using IPTG to induce the expression of recombinant proteins;
s32, centrifugally collecting thalli, performing ultrasonic crushing, and discarding bacterial lysate to obtain inclusion body sediment;
s33, fully dissolving inclusion body sediment by using inclusion body dissolving solution of urea, adjusting the concentration of inclusion body protein, dialyzing the dissolved inclusion body protein by using inclusion body renaturation buffer solution containing high-concentration urea to low-concentration urea and PBS buffer solution to obtain renaturation recombinant protein, and purifying the renaturation recombinant protein to obtain the multimeric recombinant protein.
As a further technical scheme, in the step S31, IPTG is added to a final concentration of 0.4mmol/L, the induction temperature is 37 ℃, and the induction time is 4h.
The invention also provides application of the LHRH4-CRM197-LF multimeric recombinant protein in preparing a product for regulating and controlling the reproductive capacity of mammals.
The invention also provides a vaccine comprising the LHRH4-CRM197-LF multimeric recombinant protein.
Compared with the prior art, the invention has the following advantages and beneficial effects.
1. The invention designs a novel expression vector system pGEX-LHRH4-linker1-CRM197-linker2-LF for simultaneously expressing LHRH4 polymer, recombinant diphtheria toxin nontoxic mutant CRM197 protein, recombinant lactoferrin and flexible peptide, and the LHRH4-CRM197-LF multimeric recombinant protein is obtained through expression of the expression vector, and has uniqueness and specificity in structure.
2. Under the coordination mechanism of LHRH4 polymer, recombinant diphtheria toxin nontoxic mutant CRM197 protein and recombinant lactoferrin, the multimeric recombinant protein can effectively induce the production of LHRH antibodies by orally taking or injecting the mammal, can generate more durable and obvious function of inhibiting reproductive capacity, and has important functions on reproductive regulation, hormone regulation, medical research and clinical application of the mammal.
3. In the structure of the recombinant protein, the recombinant lactoferrin has stronger proteolytic resistance, so that the recombinant protein after oral administration can keep stable in the gastrointestinal tract and promote the retention time, the absorption of the polypeptide is facilitated, more lactoferrin receptors exist in the small intestine, and the recombinant protein or the polypeptide is easier to be absorbed by the digestive tract under the mediation and transportation of the lactoferrin receptors.
4. In the multimeric recombinant protein structure, the recombinant diphtheria toxin nontoxic mutant CRM197 protein can effectively improve the drug transport effect in nerves, improve the delivery efficiency of the N-terminal LHRH4 polypeptide complex, and improve the stability of the polypeptide in vivo by the serial expression of the LHRH4 polypeptide complex and a plurality of recombinant proteins; the flexible peptide can make the recombinant protein or polypeptide spaces at two sides independent from each other, and can realize independent functions.
5. The multimeric recombinant protein has a composite structure composed of various proteins, the carried CRM197 can effectively improve the drug transport effect in nerves, the carried recombinant lactoferrin can effectively improve the oral absorption effect of the recombinant protein, compared with GnRH6 polymer-CRM 197 recombinant protein which is simultaneously expressed by a prokaryotic expression system, the recombinant protein is more stable in the gastrointestinal tract or after injection, the LHRH antibody can be more effectively induced to generate by oral administration, and the stress response of the injection to animals is avoided.
Drawings
In order to more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the drawings that are needed in the examples will be briefly described below, it being understood that the following drawings only illustrate some examples of the present invention and therefore should not be considered as limiting the scope, and that other related drawings may be obtained from these drawings without inventive effort for a person skilled in the art.
In the drawings:
FIG. 1 is a diagram showing hydrophobicity analysis of LHRH4-CRM197-LF multimeric recombinant protein of the present invention;
FIG. 2 is a diagram showing the three-level structure analysis of the LHRH4-CRM197-LF multimeric recombinant protein of the present invention;
FIG. 3 is a diagram showing the double restriction enzyme identification of the recombinant plasmid constructed according to the present invention;
FIG. 4 is a schematic diagram of the construction of a recombinant plasmid constructed in accordance with the present invention;
FIG. 5 is a graph showing the results of IPTG induction of LHRH4-CRM197-LF multimeric recombinant protein at different IPTG concentrations;
FIG. 6 is a Western-blot identification chart of the LHRH4-CRM197-LF multimeric recombinant protein of the invention;
FIG. 7 shows serum detection results after immunization of mice; wherein, graph A shows LHRH4-CRM197-LF multimeric recombinant protein immune male mouse LHRH level, graph B shows LHRH4-CRM197-LF multimeric recombinant protein immune female mouse LHRH level, and graph C shows LHRH4-CRM197-LF multimeric recombinant protein immune male mouse testosterone (T) concentration;
FIG. 8 is a color-coded image of a testis tissue section H & E of a male mouse; wherein, A diagram represents LHRH4-CRM197-LF immune male mouse testis tissue section H & E color staining detection (40X); panel B shows LHRH4-CRM197-LF immunized male mouse testis tissue sections H & E color-staining (4X); panel C shows H & E staining of tissue sections of male rat testis in normal saline (40X); d graph shows physiological saline (female) group male mouse testis tissue section H & E color staining and detecting (4X);
FIG. 9 is a graph of testicle and ovary volume comparison of immunized mice; wherein, the A diagram is LHRH4-CRM197-LF immune group testis and ovary volume diagram, the B diagram is physiological saline group testis and ovary volume diagram, the C diagram is megestrol acetate group testis and ovary volume diagram, and the D diagram is operation control group testis and ovary volume diagram;
FIG. 10 is a graph of results of different treatment groups inducing canine LHRH antibodies; wherein, graph A shows the results of physiological saline induced canine LHRH antibodies; panel B shows the canine LHRH antibody levels of LHRH4-CRM197-LF multimeric recombinant protein after 2 weeks of injection; panel C shows the canine LHRH antibody levels of LHRH4-CRM197-LF multimeric recombinant protein after 2 weeks of oral administration; panel D shows oral GnRH6-CRM197 recombinant protein canine LHRH antibody levels;
FIG. 11 is a graph showing the results of different treatment groups inducing feline LHRH antibodies; wherein, graph A shows the results of saline induced cat LHRH antibodies; panel B shows LHRH4-CRM197-LF multimeric recombinant protein levels in cats at LHRH antibody level 2 weeks after injection; panel C shows LHRH4-CRM197-LF multimeric recombinant protein levels in cats at LHRH antibody levels after 2 weeks of oral administration; panel D shows oral GnRH6-CRM197 recombinant protein cat LHRH antibody levels.
Detailed Description
For the purpose of making apparent the objects, technical solutions and advantages of the present invention, the present invention will be further described in detail with reference to the following examples and the accompanying drawings, wherein the exemplary embodiments of the present invention and the descriptions thereof are for illustrating the present invention only and are not to be construed as limiting the present invention.
The experimental materials mainly used are described below.
1. Experimental animals: c57BL/6 mice were purchased from Jiangsu Ji Yi kang biotechnology Co., ltd, beagle dogs and cats were purchased from CRO enterprises.
2. Experimental samples: ecoli, DH 5. Alpha. Competent, ecoli, BL21 (DE 3) competent, pGEX-5X-1-H (RBS) vector.
3. The main reagent comprises:
list I, list of reagents
| Reagent(s) | Manufacturer' s |
| 0.25% trypsin | Gibco Co Ltd |
| Fetal Bovine Serum (FBS) | Hyclone Co., ltd |
| Phosphate Buffer (PBS) | BEIJING SOLARBIO TECHNOLOGY Co.,Ltd. |
| PBST buffer | BEIJING SOLARBIO TECHNOLOGY Co.,Ltd. |
| TBST buffer | BEIJING SOLARBIO TECHNOLOGY Co.,Ltd. |
| isopropyl-beta-D-thiogalactoside (IPTG) | BEIJING SOLARBIO TECHNOLOGY Co.,Ltd. |
| DEPC water | Biyundian biotechnology Co.Ltd |
| Physiological saline | BEIJING SOLARBIO TECHNOLOGY Co.,Ltd. |
| Freund's complete adjuvant | Biyundian biotechnology Co.Ltd |
| Viral genome DNA/RNA extraction kit | Tiangen Biochemical technology (Beijing) Co., ltd |
| All-in-one 1 st Strand cDNA Synthesis SuperMix | Offshore protein technologies Co Ltd |
| Agarose gel DNA recovery kit | Tiangen Biochemical technology (Beijing) Co., ltd |
| PCR 2X Tag Plus PCR Master Mix (dye-containing) | Tiangen Biochemical technology (Beijing) Co., ltd |
| Plasmid small-size kit | Omega Co Ltd |
| Endonuclease enzyme | Biyundian biotechnology Co.Ltd |
| T4 DNA ligase | Neugen Biotechnology Co Ltd |
| Agar powder | BEIJING SOLARBIO TECHNOLOGY Co.,Ltd. |
| Sodium chloride (NaCl) | CHENGDU CHRON CHEMICALS Co.,Ltd. |
| Tryptone | Oxoid Co Ltd |
| Yeast extract | Oxoid Co Ltd |
| Ampicillin (Ampicilin, amp+) | BioFROXX Co |
| Coomassie brilliant blue dyeing and decolorizing set | BEIJING SOLARBIO TECHNOLOGY Co.,Ltd. |
| DNA marker | Tiangen Biochemical technology (Beijing) Co., ltd |
| Protein marker | Biyundian biotechnology Co.Ltd |
| Agarose gel | Bioweste |
| SYBR Safe DNA gel dye | Thermo Fisher Scientific |
| 50. XTAE running buffer | Bioengineering (Shanghai) Co., Ltd. |
| Urea | Chemie of Metropolis ColonProducts Co Ltd |
| Trimethylolaminomethane hydrochloride (Tris-HCl) | WUHAN SERVICEBIO TECHNOLOGY Co.,Ltd. |
| Ethylenediamine tetraacetic acid (EDTA) | Source leaf organisms |
| Sodium bicarbonate (NaHCO) 3 ） | Source leaf organisms |
| 100 mmol/L phenylmethylsulfonyl fluoride (PMSF) | Biyundian biotechnology Co.Ltd |
| Glycerol | CHENGDU CHRON CHEMICALS Co.,Ltd. |
| SDS gel preparation kit | Biyundian biotechnology Co.Ltd |
| SDS-PAGE electrophoresis buffer | Biyundian biotechnology Co.Ltd |
| 5. XSDS-PAGE protein loading buffer | Biyundian biotechnology Co.Ltd |
| Western transfer membrane liquid | Biyundian biotechnology Co.Ltd |
| HRP-labeled goat anti-rabbit IgG | British UKAbcam Co Ltd |
| HRP-labeled rabbit anti-pig IgG | BEIJING BIOSYNTHESIS BIOTECHNOLOGY Co.,Ltd. |
| Hypersensitive ECL chemiluminescence kit | Biyundian biotechnology Co.Ltd |
| BCA protein concentration determination kit | Tiangen Biochemical technology (Beijing) Co., ltd |
| ELISA coating liquid | BEIJING SOLARBIO TECHNOLOGY Co.,Ltd. |
| Skimmed milk powder | BEIJING SOLARBIO TECHNOLOGY Co.,Ltd. |
| Gelatin | BEIJING SOLARBIO TECHNOLOGY Co.,Ltd. |
| Bovine Serum Albumin (BSA) | BioFROXX Co |
| Chloroform (chloroform) | Chongqing Kogyo Chemie Co Ltd |
| Isopropyl alcohol | Chenopodium chemical reagent factory |
| Absolute ethyl alcohol | SHANGHAI MACKLIN BIOCHEMICAL Co.,Ltd. |
| Sulfuric acid (H) 2 SO 4 ） | Chenopodium chemical reagent factory |
| TMB bi-component color development liquid kit | BEIJING SOLARBIO TECHNOLOGY Co.,Ltd. |
。
4. And (5) preparing related reagents.
(1) 1% agarose gel: weighing 1.5. 1.5 g agarose, adding 100 mL of 1 xTAE electrophoresis buffer, heating to be completely melted by a microwave oven, cooling to 50-60 ℃, and adding 4 μl SYBR Safe DNA gel dye.
(2) Ampicillin (amp+) (50 μg/mL): 0.5 g of Amp+ is weighed, 8 mL is added to sterilize ddH 2 O, after being fully dissolved, the volume is fixed to 10 mL, and after being filtered by a 0.22 mu m filter membrane, the O is placed at the temperature of minus 20 ℃ for storage.
(3) LB liquid medium: 1g tryptone, 0.5 g yeast extract, 1g NaCl were weighed and 50 mL ddH was added 2 After fully stirring and dissolving O, fixing the volume to 100 mL, adjusting the pH value to 7.2-7.4, sterilizing at high temperature and high pressure, cooling and storing at 4 ℃.
(4) Amp+ resistant LB liquid medium (amp+ final concentration 100 μg/mL): 100 200 mu L of Amp+ (50 mg/mL) is added into the mL of LB liquid medium, and the mixture is fully mixed.
(5) Amp+ resistant LB solid plates: 1g tryptone, 0.5 g yeast extract, 1g NaCl,1.5 g agar powder were weighed and 50 mL ddH was added 2 And (3) fully stirring and dissolving O, then fixing the volume to 100 mL, regulating the pH value to 7.2-7.4, sterilizing at high temperature and high pressure, cooling to 50-60 ℃, adding 200 mu L of prepared Amp+ (50 mg/mL), fully and uniformly mixing, pouring into a flat plate, solidifying, and storing at 4 ℃.
(6) SDS-PAGE gel preparation:
table II, 12% separating gel formula
| Distilled water | 1.3 mL |
| 30% Acr-Bis(29:1) | 1.7 mL |
| 1 mol/L Tris（PH 8.8） | 1.9 mL |
| 10% SDS | 0.05 mL |
| 10% gel polymerization catalyst | 0.05 mL |
| TEMED | 0.002 mL |
| 5 mL |
Table III, 5% concentrated glue formulation
| Distilled water | 1.4 mL |
| 30% Acr-Bis(29:1) | 0.33 mL |
| 1 mol/L Tris（PH 6.8） | 0.25 mL |
| 10% SDS | 0.02 mL |
| 10% gel polymerization catalyst | 0.02 mL |
| TEMED | 0.002 mL |
| 2 mL |
。
(7) Western blot blocking solution: 5 g of BSA was weighed out and added to 100 mL of TBST buffer, and the mixture was dissolved with stirring.
(8) Proteolytic renaturation related reagent.
(1) Ultrasonic disruption buffer: tris-HCl 15.76 g,EDTA 14.6 g,NaCl 29.22 g was weighed and 500 mL ddH was added 2 O is fully stirred and dissolved, 50 mL glycerol is added, the volume is fixed to 1000 mL, and the mixture is preserved at 4 ℃.
(2) Inclusion body wash I: tris-HCl 1.58 g,EDTA 2.92 g, urea 12.12 g are weighed, 200 mu L TritonX-100,1,000 mL ddH are added 2 And (5) fully stirring and dissolving O, regulating the pH value to 8.0,4 ℃ and preserving.
(3) Inclusion body wash II: tris-HCl 1.58 g,EDTA 2.92 g was weighed and 200. Mu.L TritonX-100,1,000 mL ddH was added 2 And (5) fully stirring and dissolving O, regulating the pH value to 8.0,4 ℃ and preserving.
(4) Inclusion body dissolution Buffer a (for use at present): weighing 0.25 g of Tris-HCl and NaCl 2.34 g, urea 38.44 g, added 80 mL ddH 2 And (5) fully stirring and dissolving O, regulating the pH value to 8.0,4 ℃ and preserving.
(5) Dialysis bag treatment fluid I: weighing NaHCO 3 10 g, EDTA 0.15. 0.15 g, 490 mL ddH was added 2 And (5) fully stirring and dissolving O, regulating the pH value to 8.0,4 ℃ and preserving.
(6) Dialysis bag treatment liquid II: EDTA 0.15. 0.15 g was weighed and 500 mL ddH was added 2 And (5) fully stirring and dissolving O, regulating the pH value to 8.0,4 ℃ and preserving.
(7) Dialysis Buffer B (as prepared): tris-HCl 0.63 g,NaCl 5.84 g, urea 72.07 g were weighed and 0.8 mL 100 mmol/L PMSF and 200mL ddH were added 2 And (5) fully stirring and dissolving O, regulating the pH value to 8.0,4 ℃ and preserving.
(8) Dialysis Buffer C (as-prepared): tris-HCl 0.63 g,NaCl 5.84 g, urea 48.05 g were weighed and added with 0.8 mL 100 mmol/L PMSF and 200mL ddH 2 And (5) fully stirring and dissolving O, regulating the pH value to 8.0,4 ℃ and preserving.
(9) Dialysis Buffer D (as-prepared): tris-HCl 0.63 g,NaCl 5.84 g, urea 24.02 g were weighed and 0.8 mL 100 mmol/L PMSF and 200mL ddH were added 2 And (5) fully stirring and dissolving O, regulating the pH value to 8.0,4 ℃ and preserving.
Dialysis Buffer E: tris-HCl 0.63 g,NaCl 5.84 g was weighed and 0.8 mL of 100 mmol/L PMSF and 200mL of ddH were added 2 And (5) fully stirring and dissolving O, regulating the pH value to 8.0,4 ℃ and preserving.
Example 1.
The embodiment provides an LHRH4-CRM197-LF multimeric recombinant protein, which has an amino acid sequence shown as SEQ ID NO. 2.
Example 2.
The embodiment provides a preparation method of LHRH4-CRM197-LF multimeric recombinant protein, which specifically comprises the following steps:
1. gene design encoding multimeric recombinant proteins.
According to LHRH4 polymer (SEQ ID NO. 3), flexible peptide 1 sequence (SEQ ID NO. 4), recombinant CRM197 protein (SEQ ID NO. 5), flexible peptide 2 sequence (SEQ ID NO. 6) and recombinant lactoferrin sequence (SEQ ID NO. 7), a nucleotide sequence with the total length of 4011bp shown as SEQ ID NO.1 is designed and obtained for cloning into an enzyme cutting site of a vector.
2. Construction of recombinant plasmid pGEX-LHRH4-linker-CRM 197-linker-LF.
The invention selects pGEX-5X-1-H (RBS) vector as the basic skeleton structure of the vector, and the expression vector of the recombinant protein can be constructed by using other known prokaryotic expression vectors.
2.1 vector extraction: mu.g pGEX-5X-1-H (RBS) (from Kirschner Biotechnology Co., ltd.) was added to 100. Mu.L DH 5. Alpha. Competent cells, and the tubes were thoroughly mixed and then ice-bathed for 30min. After ice bath, the competent cells were incubated at 42℃for 90s and ice-bathed again for 2min. 800. Mu.L of LB medium was added to the centrifuge tube, and the culture was performed at 37℃for 1h with shaking at 200 rpm/min. 10-50 mu L of transformed competent cells were plated on Amp dishes, cultured at 37℃for 12-24 hours, single colonies were picked up and expanded in 200mL LB medium (ampicillin was added to 100. Mu.g/mL), and pGEX-5X-1-H (RBS) plasmids were extracted using QIAGEN plasmid miniprep kit (QIAGEN, germany).
2.2 enzyme digestion: use carrier double enzyme system BamHI enzyme 0.5 [ mu ] L, ecoRI enzyme 0.5 [ mu ] L, pGEX-5X-1-H (RBS) carrier 6 [ mu ] g, 10xBuffer R2 [ mu ] L, ddH 2 O10 mu L, and carrying out constant-temperature double enzyme digestion for 2h at 37 ℃. Double enzyme digestion followed by electrophoresis on a 1% agarose gel and purification of the digested pGEX-5X-1-H (RBS) using a QIAGEN DNA gel recovery kit.
2.3 ligation, transformation and expansion culture: pGEX-5X-1-H (RBS) 3 [ mu ] g after enzyme digestion, 7 [ mu ] g of nucleotide sequence shown in SEQ ID NO.1, 1 [ mu ] g of T4 DNA ligase and 10xBuffer R2 [ mu ] L, ddH 2 O7 mu L, and connecting overnight at 4 ℃ after being fully mixed in a centrifuge tube, so that a nucleotide sequence shown in SEQ ID NO.1 with the full length of 4011bp is cloned into a BamHI/EcoRI digestion site of a pGEX-5X-1 vector, then, after a connection product is transformed into E.coli DH5 alpha competent cells, E.coli DH5 alpha is subjected to expansion culture, and a QIAGEN plasmid miniextraction kit is used for extracting recombinant plasmids. And the recombinant plasmid is identified by BamHI/EcoRI double enzyme digestion identification and second generation sequencing, and the identification is correctThe recombinant plasmid of (a) is named:
pGEX-LHRH4-linker1-CRM197-linker2-LF。
expression and purification of LHRH4-CRM197-LF multimeric recombinant protein.
The recombinant plasmid pGEX-LHRH4-linker1-CRM197-linker2-LF is transformed into E.coli BL21 gene engineering bacteria, and the BL21 engineering bacteria after plasmid transformation is named BL21-LHRH4-linker1-CRM197-linker2-LF. The bacteria were cultured in a shaking manner at 200rpm to a logarithmic growth phase (OD 600nm of about 0.6 to 0.8) at 37℃and BL21 was treated with IPTG at a concentration of 0.2mM for 4 hours to induce expression of the recombinant protein.
And (3) centrifuging at 3000rpm for 10min to collect thalli, carrying out ultrasonic crushing on the collected thalli, separating bacterial thalli from bacterial lysis supernatant, and discarding the supernatant to obtain inclusion body sediment. And (3) fully dissolving inclusion body sediment by using an inclusion body dissolving solution of 8 mol/L urea, adjusting the concentration of inclusion body protein, and dialyzing the dissolved inclusion body protein by using an inclusion body renaturation buffer solution containing high-concentration urea to low-concentration urea and a PBS buffer solution to obtain renaturation protein. The renatured recombinant protein was purified using a GST-tagged protein purification kit. And finally, performing SDS-PAGE electrophoresis and Western-blot analysis on the bacterial lysate, bacterial lysate supernatant and purified recombinant protein respectively. And the recombinant protein was named: LHRH4-CRM197-LF multimeric recombinant protein.
Example 3.
This embodiment differs from embodiment 2 in that: in the expression and purification process of the recombinant multimeric protein, BL21 was treated with IPTG at a concentration of 0.4. 0.4mM for 4h.
Example 4.
This embodiment differs from embodiment 2 in that: in the process of expression and purification of the multimeric recombinant protein, BL21 was treated with IPTG at a concentration of 0.8. 0.8 mM for 4h.
The following are the relevant detection results of recombinant plasmids and multimeric recombinant proteins prepared in example 3.
1. Results of bioinformatics analysis of multimeric recombinant proteins.
The results of the on-line software Antibody Epitope Prediction analysis of the antigen epitope region of the LHRH4-CRM197-LF recombinant protein showed that the amino acid positions of 5-154, 179-206, 282-357, 364-405, 449-469, 471-493, 570-586, 610-623, 746-768, 782-793, 921-968, 973-986, 999-1011, 1062-1075, 1087-1111, 1123-1146, 1151-1171, 1193-1213, 1220-1231, 1265, 1281, 1295-1308 were dominant antigen epitope regions (see Table IV). The polymeric recombinant protein is described as having a structural basis for inducing specific antibodies.
Table IV, LHRH4-CRM197-LF multimeric recombinant protein epitope analysis results
| Sequence number | Starting point | Endpoint (endpoint) | Polypeptide sequence | Length of |
| 1 | 5 | 154 | KREHWSYGLRPGG KREHWSYGLRPGG KREHWSYGLRPGG KREHWSYGLRPGG KRKLGGGGSGGGG SGGGGSMGADDVV DSSKSFVMENFSS YHGTKPGYVDSIQ KGIQKPKSGTQGN YDDDWKEFYSTDN KYDAAGYSVDNEN PLSGKAG | 150 |
| 2 | 177 | 177 | T | 1 |
| 3 | 179 | 206 | KKELGLSLTEPLM EQVGTEEFIKRFG DG | 28 |
| 4 | 217 | 233 | AEGSSSVEYINNW EQAK | 17 |
| 5 | 244 | 253 | ETRGKRGQDA | 10 |
| 6 | 264 | 277 | GNRVRRSVGSSLS C | 14 |
| 7 | 282 | 357 | WDVIRDKTKTKIE SLKEHGPIKNKMS ESPNKTVSEEKAK QYLEEFHQTALEH PELSELKTVTGTN PVFAGANYAAW | 76 |
| 8 | 364 | 405 | VIDSETADNLEKT TAALSILPGIGSV MGIADGAVHHNTE EIV | 42 |
| 9 | 449 | 469 | NSYNRPAYSPGHK TQPFLHDG | 21 |
| 10 | 471 | 493 | AVSWNTVEDSIIR TGFQGESGHD | 23 |
| 11 | 514 | 527 | PGKLDVNKSKTHI S | 14 |
| 12 | 541 | 542 | DG | 2 |
| 13 | 544 | 547 | VTFC | 4 |
| 14 | 552 | 557 | PVYVGN | 6 |
| 15 | 570 | 586 | SSSEKIHSNEISS DSIG | 17 |
| 16 | 590 | 599 | YQKTVDHTKV | 10 |
| 17 | 610 | 623 | KSGGGGSGGGGSG G | 14 |
| 18 | 648 | 648 | R | 1 |
| 19 | 661 | 665 | WFKCR | 5 |
| 20 | 669 | 677 | WRMKKLGAP | 9 |
| 21 | 714 | 717 | RDPY | 4 |
| 22 | 730 | 731 | KE | 2 |
| 23 | 746 | 768 | GSNFQLDQLQGRK SCHTGLGRSA | 23 |
| 24 | 782 | 793 | SWTESLEPLQGA | 12 |
| 25 | 797 | 797 | F | 1 |
| 26 | 799 | 800 | SA | 2 |
| 27 | 803 | 812 | VPCIDRQAYP | 10 |
| 28 | 821 | 835 | EGENQCACSSREP YF | 15 |
| 29 | 859 | 869 | VFENLPEKADR | 11 |
| 30 | 878 | 887 | NNSRAPVDAF | 10 |
| 31 | 906 | 910 | DGKED | 5 |
| 32 | 921 | 968 | EKSGKNKSRSFQL FGSPPGQRDLLFK DSALGFLRIPSKV DSALYLGSR | 48 |
| 33 | 971 | 971 | T | 1 |
| 34 | 973 | 986 | LKNLRETAEEVKA R | 14 |
| 35 | 999 | 1011 | EQKKCQQWSQQSG | 13 |
| 36 | 1044 | 1047 | IYTA | 4 |
| 37 | 1062 | 1075 | SSKHSSLDCVLRP T | 14 |
| 38 | 1087 | 1111 | ANEGLTWNSLKDK KSCHTAVDRTAG | 25 |
| 39 | 1123 | 1146 | TGSCAFDEFFSQS CAPGADPKSRL | 24 |
| 40 | 1151 | 1171 | AGDDQGLDKCVPN SKEKYYGY | 21 |
| 41 | 1193 | 1213 | VWENTNGESTADW AKNLNRED | 21 |
| 42 | 1220 | 1231 | DGTRKPVTEAQS | 12 |
| 43 | 1265 | 1281 | KNGKNCPDKFCLF KSET | 17 |
| 44 | 1291 | 1293 | ECL | 3 |
| 45 | 1295 | 1308 | KLGGRPTYEEYLG T | 14 |
| 46 | 1320 | 1323 | CSTS | 4 |
| 47 | 1331 | 1332 | FL | 2 |
。
1.2, the LHRH4-CRM197-LF multimeric recombinant protein is subjected to basic physicochemical property analysis by using on-line software ProtParam, and the result shows that the molecular weight of the LHRH4-CRM197-LF multimeric recombinant protein is 145573.91, and the theoretical isoelectric point (isoelectric point, pI) is 8.48. The half-lives of LHRH4-CRM197-LF recombinant protein in reticulocytes (in vitro), yeast cells and coliform bacteria are respectively 30h, >20h, >10h, and the fat solubility index is 75.93.
1.3, the hydrophobicity of the LHRH4-CRM197-LF multimeric recombinant protein was analyzed by using the on-line software ProtScale (see FIG. 1), and the result shows that the protein has certain hydrophobicity, the maximum hydrophobicity index at 633 amino acids is 2.4, and the minimum hydrophobicity index at 869 amino acids is-2.822.
1.4 analysis of LHRH4-CRM197-LF multimeric recombinant protein tertiary structure was performed using the on-line software SWISS-MODEL software (see FIG. 2). The results show that the analysis results show that the LHRH4, the CRM197 and the LF can be combined by the flexible peptide, which is beneficial to the independent biological activity of each part of the recombinant protein.
1.5, predicting a LHRH4-CRM197-LF multimeric recombinant protein transmembrane region by using online software TMHMMP, and displaying that the recombinant protein does not have a transmembrane region structure, which indicates that the recombinant protein does not belong to membrane proteins and is beneficial to expression and mass production of the recombinant protein.
2. And (5) identifying recombinant plasmids.
As shown in FIG. 3, a double restriction enzyme identification chart of recombinant plasmids is shown, wherein M represents a DNA Marker band, and 1 represents a recombinant plasmid band before restriction enzyme; 2 shows the recombinant plasmid band after enzyme digestion, and as can be seen from FIG. 3, the recombinant plasmid has a target band at 4011bp through double enzyme digestion identification and has correct sequencing result, which indicates that the recombinant plasmid is successfully constructed, and FIG. 4 is a schematic diagram of the recombinant plasmid structure constructed by the invention, and is named pGEX-LHRH4-linker1-CRM197-linker2-LF.
3. Expression and identification of LHRH4-CRM197-LF multimeric recombinant protein.
Experimental results show that after IPTG induction, the LHRH4-CRM197-LF multimeric recombinant protein is stably expressed in E.coli BL21 (DE 3) genetic engineering bacteria, the molecular weight is 145.6kDa, the biological informatics prediction result is met, and the LHRH4-CRM197-LF multimeric recombinant protein is mainly expressed in the form of inclusion bodies.
As shown in FIG. 5, the IPTG induction results of the recombinant multimeric proteins at different IPTG concentrations are shown to be 0.4mmol/L, the induction temperature is 37 ℃, and the induction time is 4h, which is the optimal induction condition of the recombinant multimeric proteins.
As shown in FIG. 6, the Western-blot identification of the LHRH4-CRM197-LF multimeric recombinant protein shows that the purified LHRH4-CRM197-LF multimeric recombinant protein can be combined with an LHRH specific antibody, and the purified recombinant protein has biological activity and can be applied to subsequent production and clinical application.
The following is a verification of the effect of the LHRH4-CRM197-LF recombinant multimeric protein prepared in example 3 on the fertility inhibiting ability and the ability to induce LHRH antibody production, including control experiments.
1. Mice immunization experiments.
1.1, experimental and control designs.
C57BL/6 male mice were divided into: LHRH4-CRM197-LF treatment (12), normal saline (12), megestrol acetate (12), surgical sterilization (12), gnRH6-CRM197 recombinant protein treatment (12) control.
C57BL/6 master mice were divided into: LHRH4-CRM197-LF female (12), normal saline female (12), ethinyl estradiol female (12), surgical sterilization female (12), gnRH6-CRM197 recombinant protein treatment female control (12).
1.2 immunization protocol.
On week 0, LHRH4-CRM197-LF, control GnRH 6-mer-CRM 197 recombinant protein was subcutaneously injected into mice at 1 μg recombinant protein per 1g body weight per mouse. Megestrol acetate is fed to the megestrol acetate group according to the weight of 0.1 mu g/1g, and ethinyl estradiol is fed to the ethinyl estradiol group according to the weight of 0.1 mu g/1 g. On week 2, LHRH4-CRM197-LF, control GnRH6 mer-CRM 197 recombinant protein was subcutaneously injected into treated mice at 1 μg recombinant protein per 1g body weight per mouse.
All experimental mice were collected at week 0 (pre-immunization), week 3, week 6, and week 9, and serum LHRH antibody, testosterone (Testosterone, T) levels were measured by ELISA. At week 9, 4 mice were randomly selected in each group for histologic sectioning of testes and ovaries.
The following mice experimental groups were pair-fed at week 10: the female mice of the LHRH4-CRM197-LF treatment male group and the female mice of the LHRH4-CRM197-LF treatment female group are respectively matched and bred; pairing and feeding normal saline female mice and normal saline male mice and residual male mice of female mice respectively; the megestrol acetate female group and the ethinyl estradiol female group residual male and female mice are respectively paired and raised; pairing and feeding the remaining male mice and female mice of the surgical sterilization female group respectively; the control GnRH6 polymer-CRM 197 recombinant protein female mice and the remaining male and female mice of the female group are respectively matched and fed. And the reproduction of each group of newborn mice was observed.
The laboratory animals adopt commercial daily ration and drinking water, the temperature of the laboratory mice house is controlled at 21 ℃, the humidity is controlled at 50%, and the daily illumination/darkness duration is 12h/12h.
1.3, experimental results.
(1) The serum detection results after mice immunization are shown in fig. 7, wherein, graph A shows LHRH4-CRM197-LF multimeric recombinant protein immune male mice LHRH level, graph B shows LHRH4-CRM197-LF multimeric recombinant protein immune female mice LHRH level, and graph C shows LHRH4-CRM197-LF multimeric recombinant protein immune male mice testosterone (T) concentration.
As can be seen from FIG. 7, the use of the LHRH4-CRM197-LF multimeric recombinant protein in both C57BL/6 male and female mice effectively induced LHRH-specific antibody production and maintained higher antibody levels for 3 to 9 weeks after immunization. But neither megestrol acetate nor ethinyl estradiol has a significant effect on LHRH antibody levels.
(2) Randomly selecting 4 mice in each group at 9 weeks after immunization for histologic section microscopy of testis and ovary.
FIG. 8 is a graph of H & E staining microscopy of testis tissue sections of male mice, wherein graph A shows H & E staining microscopy (40X) of LHRH4-CRM197-LF immunized testis tissue sections of male mice; panel B shows LHRH4-CRM197-LF immunized male mouse testis tissue sections H & E color-staining (4X); panel C shows H & E staining of tissue sections of male rat testis in normal saline (40X); panel D shows H & E staining of tissue sections of testis of male mice in saline (4X).
From fig. 8, H & E staining of testis tissue sections showed that LHRH4-CRM197-LF multimeric recombinant protein immunized mice testis Qu Jingxi had a shrinking and reduced number of fine tubes, a shrinking and reduced number of mesenchymal cells, a reduced sperm and a significant degeneration of spermatogenic cells. And the level of each grade of sperm cells in the testis yeast seminiferous tubule of the normal saline group mouse is clear, and the outlines of the sperm cells, primary spermatocyte, sperm cells and interstitial cells in the testis interstitial tissue are clear and full.
Fig. 9 is a testicle and ovary volume comparison diagram of an immunized mouse, wherein, a diagram A is a testicle and ovary volume diagram of an LHRH4-CRM197-LF immunized group, a diagram B is a testicle and ovary volume diagram of a physiological saline group, a diagram C is a testicle and ovary volume diagram of an megestrol acetate group, and a diagram D is a testicle and ovary volume diagram of an operation control group.
From fig. 9, it can be seen that LHRH4-CRM197-LF multimeric recombinant protein immunized mice have testes and ovaries with significantly lower volumes than those of the B saline group, the C chemical group, and the D surgery control group, indicating that the multimeric recombinant protein can significantly inhibit reproductive function.
(3) Mice were pair fed according to the immunization protocol of 1.2 at week 10 post immunization, and the reproduction results were observed 28 weeks after the first immunization, and the results showed that: LHRH4-CRM197-LF multimeric recombinant protein immunized male and female mice and surgical sterilization male and female mice the number of breeding newborn mice was 0; the total number of the reproduction of the normal saline mice is 139; the total reproduction number of megestrol acetate female groups and ethinyl estradiol female groups is 108; the control GnRH6 mer-CRM 197 recombinant protein treated group propagated a total of 64.
The results show that the LHRH4-CRM197-LF multimeric recombinant protein designed by the invention has obvious reproduction inhibition effect on C57BL/6 reproduction capacity within 28 weeks after immunization, and the inhibition effect within 28 weeks of the first immunization is obviously stronger than that of megestrol acetate, ethinyl estradiol and control GnRH6 polymer-CRM 197 recombinant protein, and has longer reproduction inhibition effect compared with GnRH6 polymer-CRM 197 recombinant protein.
In conclusion, through a mouse immunization experiment, the LHRH antibody can be effectively induced to generate after mice are immunized by injection, testosterone secretion is inhibited, and the reproduction capability of the mice is continuously inhibited within 28 weeks after immunization, so that the LHRH4-CRM197-LF multimeric recombinant protein has obvious and durable reproduction inhibiting effect, and has better reproduction inhibiting capability compared with the GnRH6 polymer-CRM 197 recombinant protein which is simultaneously expressed through a prokaryotic expression system.
2. Dog immunization experiments.
2.1, experimental and control designs.
The 10 month old beagle (male) is divided into LHRH4-CRM197-LF injection treatment (4), multimeric recombinant protein oral treatment (4), physiological saline (4) and GnRH6 polymer-CRM 197 recombinant protein oral administration (control).
2.2, immunization protocol.
At week 0, LHRH4-CRM197-LF injection treatment female group was subcutaneously injected with 1 μg recombinant protein per 1g body weight per dog; oral treatment of LHRH4-CRM197-LF, control GnRH6 Polymer-CRM 197 recombinant protein oral treatment groups were treated orally at 2 μg recombinant protein per 1g body weight per dog. At week 2, LHRH4-CRM197-LF injection treatment female groups were subcutaneously injected at 1 μg recombinant protein per 1g body weight per dog; oral treatment of LHRH4-CRM197-LF, control GnRH6 Polymer-CRM 197 recombinant protein oral treatment groups were treated orally at 2 μg recombinant protein per 1g body weight per dog. All experimental dogs were collected at week 0 (pre-immunization), weeks 2, 4, 6, 8, 10, and serum LHRH antibody levels were checked by ELISA.
2.3, experimental results.
FIG. 10 is a graph showing results of canine LHRH antibody induction by saline in different treatment groups, wherein graph A shows results of canine LHRH antibody induction; panel B shows the canine LHRH antibody levels of LHRH4-CRM197-LF multimeric recombinant protein after 2 weeks of injection; panel C shows the canine LHRH antibody levels of LHRH4-CRM197-LF multimeric recombinant protein after 2 weeks of oral administration; panel D shows oral GnRH6-CRM197 recombinant protein canine LHRH antibody levels.
As can be seen from fig. 10, physiological saline can not induce the canine LHRH antibody, the LHRH4-CRM197-LF multimeric recombinant protein can effectively induce the production of the LHRH antibody both after injection and oral immunization of dogs, while oral GnRH6-CRM197 recombinant protein does not significantly regulate the canine LHRH antibody level.
3. Cat immunization experiments.
3.1, experimental and control designs.
The cats (males) at 10 months of age were divided into LHRH4-CRM197-LF injection treatment (4), LHRH4-CRM197-LF oral treatment (4), physiological saline (4), gnRH 6-mer-CRM 197 recombinant protein oral control group.
3.2, immunization protocol: at week 0, LHRH4-CRM197-LF injection treatment female group was subcutaneously injected at 1 μg recombinant protein per 1g body weight per cat; oral treatment of LHRH4-CRM197-LF, control GnRH6 Polymer-CRM 197 recombinant protein oral treatment groups were treated orally at 2 μg recombinant protein per 1g body weight per cat. At week 2, LHRH4-CRM197-LF injection treatment female groups were subcutaneously injected at 1 μg recombinant protein per 1g body weight per cat; oral treatment of LHRH4-CRM197-LF, control GnRH6 Polymer-CRM 197 recombinant protein oral treatment groups were treated orally at 2 μg recombinant protein per 1g body weight per cat. All experimental cats were collected at week 0 (pre-immunization), weeks 2, 4, 6, 8, 10, and serum LHRH antibody levels were checked by ELISA.
3.3, experimental results.
FIG. 11 is a graph showing results of different treatment groups inducing feline LHRH antibodies, wherein graph A shows results of saline-induced feline LHRH antibodies; panel B shows LHRH4-CRM197-LF multimeric recombinant protein levels in cats at LHRH antibody level 2 weeks after injection; panel C shows LHRH4-CRM197-LF multimeric recombinant protein levels in cats at LHRH antibody levels after 2 weeks of oral administration; panel D shows oral GnRH6-CRM197 recombinant protein cat LHRH antibody levels.
As can be seen from FIG. 11, physiological saline can not induce LHRH antibody in cats, and the multimeric recombinant protein can effectively induce LHRH antibody production both after injection and oral immunization of cats, while oral GnRH6-CRM197 recombinant protein does not significantly regulate the levels of LHRH antibody in cats.
Finally, it should be noted that the LHRH multimer in the multimeric recombinant protein of the present invention may be designed into LHRH 1-12 multimer according to clinical application requirements, and the structures of different protein compositions may be adjusted and combined according to specific requirements, for example, LF and CRM197 may be replaced by other protein combinations, or CRM197-linker-LF may be replaced by a single protein as a whole, or the N-terminal to C-terminal positions of the multimeric recombinant protein may be adjusted.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the scope of the invention, but to limit the invention to the particular embodiments, and any modifications, equivalents, improvements, etc. that fall within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (8)
1. The LHRH4-CRM197-LF multimeric recombinant protein is characterized in that the gene encoding the LHRH4-CRM197-LF multimeric recombinant protein is shown as a nucleotide sequence shown as SEQ ID NO. 1.
2. A method for preparing LHRH4-CRM197-LF multimeric recombinant protein, which is characterized by comprising the following steps:
s1, designing and obtaining a nucleotide sequence shown as SEQ ID NO. 1;
s2, constructing recombinant plasmids: connecting a nucleotide sequence shown in SEQ ID NO.1 with a vector subjected to double enzyme digestion, converting the connected product into E.coli DH5 alpha competent cells, and performing amplification culture on the E.coli DH5 alpha to extract recombinant plasmids;
s3, expression and purification of a multimeric recombinant protein: the recombinant plasmid is transformed into genetic engineering bacteria, expression of the multimeric recombinant protein is induced by IPTG, and the multimeric recombinant protein of LHRH4-CRM197-LF is obtained through separation and purification.
3. The method for preparing the LHRH4-CRM197-LF multimeric recombinant protein according to claim 2, wherein the construction of the recombinant plasmid in step S2 comprises:
s21, extracting pGEX-5X-1-H plasmid;
s22, performing double enzyme digestion by using a double enzyme digestion system BamHI enzyme and EcoRI enzyme to obtain digested pGEX-5X-1-H;
s23, connecting the digested pGEX-5X-1-H with a nucleotide sequence shown in SEQ ID NO.1 under the action of T4 DNA ligase to obtain a connection product;
s24, after the connection product is transformed into E.coli DH5 alpha competent cells, E.coli DH5 alpha is subjected to expansion culture, and recombinant plasmids are extracted.
4. A method for preparing LHRH4-CRM197-LF recombinant multimeric protein according to claim 3, wherein step S22 comprises:
BamHI enzyme 0.5uL, ecoRI enzyme 0.5uL, pGEX-5X-1-H vector 6ug, 10xBuffer R2uL,ddH were used as a vector double cleavage system 2 O10 uL was double digested at 37℃for 2 hours, and after double digested pGEX-5X-1-H was purified by agarose gel electrophoresis.
5. The method for preparing the LHRH4-CRM197-LF recombinant multimeric protein according to claim 2, wherein the step S3 of expressing and purifying the recombinant multimeric protein comprises:
s31, transforming the recombinant plasmid into E.coli BL21 gene engineering bacteria, culturing the bacteria to a logarithmic growth phase in an oscillating way, and treating the bacteria by using IPTG to induce the expression of recombinant proteins;
s32, centrifugally collecting thalli, performing ultrasonic crushing, and discarding bacterial lysate to obtain inclusion body sediment;
s33, fully dissolving inclusion body sediment by using inclusion body dissolving solution of urea, adjusting the concentration of inclusion body protein, dialyzing the dissolved inclusion body protein by using inclusion body renaturation buffer solution containing high-concentration urea to low-concentration urea and PBS buffer solution to obtain renaturation recombinant protein, and purifying the renaturation recombinant protein to obtain LHRH4-CRM197-LF multimeric recombinant protein.
6. The method for preparing a recombinant protein of LHRH4-CRM197-LF according to claim 5, wherein IPTG is added to a final concentration of 0.4mmol/L in step S31, the induction temperature is 37℃and the induction time is 4h.
7. Use of LHRH4-CRM197-LF multimeric recombinant proteins according to claim 1 in the preparation of a product for modulating reproductive capacity in mammals.
8. A vaccine comprising the LHRH4-CRM197-LF multimeric recombinant protein of claim 1.
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