CN111978414A - Application of duck interferon gamma fusion protein in preparation of medicine for preventing and treating duck viral hepatitis - Google Patents
Application of duck interferon gamma fusion protein in preparation of medicine for preventing and treating duck viral hepatitis Download PDFInfo
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- 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
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
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- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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Abstract
The invention discloses application of duck interferon gamma fusion protein in preparing a medicine for preventing and treating duck viral hepatitis, wherein the duck interferon gamma fusion protein ELP-DuIFN gamma is a fusion protein obtained by fusing an elastin-like polypeptide ELP and a protein amino terminal of duck interferon gamma. The expressed ELP-DuIFN gamma is safe and nontoxic, has long half-life period and stronger antiviral activity in vivo and in vitro, and can be further researched as a backup biological medicament for preventing and treating duck viral hepatitis.
Description
Technical Field
The invention belongs to the technical field of veterinary drugs, and particularly relates to an antiviral effect of duck interferon gamma fusion protein on duck hepatitis virus 1, so as to prepare a drug for resisting the duck hepatitis virus 1.
Background
China is the first country of production and consumption of waterfowls in the world, and the large-scale duck farms are continuously and steadily increasing in recent years, but the viral infectious diseases of ducks are a big problem which troubles the duck breeding industry[1]. Duck Viral Hepatitis (DVH) is one of important epidemic diseases causing death of ducklings, the pathogeny of the DVH is duck hepatitis virus type 1 (DHAV-1), the ducklings within 4 weeks are mainly attacked, and the death rate can reach 90%. Immunization is the main method for preventing and treating DHAV-1 infection, but the occurrence of variant strains reduces the immune efficiency and even leads to immune failure, so that a new method for preventing and treating DVH is urgently needed to be searched[2]。
Interferons (IFNs) are a class of viruses with a broad spectrum of antiviral activity[3-5]Glycoprotein with multiple biological functions of resisting tumor, regulating immunity and the like[6]Mainly comprises type I and type II, and gamma interferon is type II interferon. 1999, Schultz[7]The DuIFN-gamma open reading frame is proved to contain 495 basic groups, can code 164 amino acids and has various strong biological functions of resisting virus and the like[8]. However, the source of natural DuIFN-gamma is limited, and recombinant interferon has the disadvantages of low expression level, difficult purification, short half-life, low activity and the like, and particularly, the disadvantage of short half-life limits the subsequent development of DuIFN gamma. Polyethylene glycol (PEG) method and Human Serum Albumin (HSA) combination method, and the two methods for prolonging half-life period have been used for preparing recombinant human IFN and are clinically applied[9-11]. However, pegylation has the disadvantages of low efficiency, poor clinical efficacy, obvious side effects and the like[12]Although the Human Serum Albumin (HSA) binding method has good therapeutic effect and little side effect, the price is high[13,14]。
Elastin-like polypeptides (ELPs) are pentapeptide polymers synthesized from Elastin repeats in vivo[15]Has the temperature-sensitive reversible phase transition characteristic, and is commonly used for protein purification and drug delivery due to low immunogenicity and good biocompatibility[16]. Therefore, the research applies ELP to the purification and half-life extension of DuIFN gamma, and develops the research of the antiviral effect of ELP-DuIFN gamma on DHAV-1.
Disclosure of Invention
Aiming at the technical problems brought forward by the background technology, the invention aims to provide duck interferon gamma fusion protein ELP-DuIFN gamma and application thereof.
The invention also aims to provide a duck interferon gamma fusion protein ELP-DuIFN gamma expression vector.
The invention also aims to provide a duck interferon gamma gene optimized by codons.
The purpose of the invention can be realized by the following technical scheme:
a duck interferon gamma fusion protein ELP-DuIFN gamma is a fusion protein obtained by fusing an elastin-like polypeptide ELP and the protein amino terminal of duck interferon gamma.
The preparation method of the duck interferon gamma fusion protein ELP-DuIFN gamma comprises the following steps: the synthesis of the amino acid sequence shown as SEQ ID NO: 1 and introducing SacI and XhoI enzyme cutting sites at two ends, inserting the DuIFN gamma gene between the SacI and XhoI enzyme cutting sites of pET30a-ELP vector, screening to obtain a recombinant vector pET30a-ELP-DuIFN gamma, converting the recombinant vector pET30a-ELP-DuIFN gamma into a competent expression strain to obtain an expression engineering bacterium, and performing induction expression, protein purification and endotoxin removal to obtain a fusion protein ELP-DuIFN gamma.
A codon optimized duck interferon gamma gene has a nucleotide sequence shown as SEQ ID NO. 1.
A duck interferon gamma fusion protein ELP-DuIFN gamma expression vector, the construction method of the expression vector is: the synthesis of the amino acid sequence shown as SEQ ID NO: 1 and introducing SacI and XhoI enzyme cutting sites at two ends, inserting the DuIFN gamma gene between the SacI and XhoI enzyme cutting sites of pET30a-ELP vector, and screening to obtain recombinant vector pET30a-ELP-DuIFN gamma.
A transgenic cell line or an expression engineering bacterium containing the duck interferon gamma fusion protein ELP-DuIFN gamma expression vector.
The application of the duck interferon gamma fusion protein ELP-DuIFN gamma in preparing the medicine for preventing and treating duck viral hepatitis.
The application of the duck interferon gamma fusion protein ELP-DuIFN gamma in preparing the medicine for resisting the duck hepatitis 1 virus. The administration mode of the medicine is drench administration.
The invention has the beneficial effects that:
the fusion protein ELP-DuIFN gamma of the invention is safe and nontoxic, has long half-life period, has stronger antiviral activity in vivo and in vitro, and can be further researched as a backup biological medicament for preventing and treating duck viral hepatitis.
Drawings
FIG. 1 shows the restriction enzyme identification of pET30a-ELP-DuIFN gamma; wherein, M is 1kb DNA Ladder; 1 Xho I/SacI double diagnostic of pET30a-ELP-DuIFN γ.
FIG. 2 is an ELP-DuIFN γ expression and purification SDS-PAGE analysis; wherein, M: protein Marker; 1: ELP-DuIFN gamma purified protein; 2: inducing the supernatant of the recombinant bacteria; 3: inducing the precipitation of recombinant bacteria; 4: empty vector pET30-ELP control; 5: ITC primary purification of the protein.
FIG. 3 is an SDS-PAGE identification of ELP-DuIFN α after purification; wherein, M: protein Marker; 1: purified ELP-DuIFN α.
FIG. 4 is an in vitro half-life analysis of ELP-DuIFN γ.
FIG. 5 is an in vivo half-life analysis of ELP-DuIFN γ.
FIG. 6 shows the results of flow cytometry; wherein: the upper left quadrant indicates positive CD8, the lower right quadrant indicates positive CD4, and PerCP-Cy5.5 and APC are fluorescein.
FIG. 7 shows different groups at different times CD4+/CD8+Comparing the ratio; wherein: RT: room temperature (about 25 ℃); w: week.
FIG. 8 is a comparison of the change in ELP-DuIFN γ content at different storage temperatures; wherein: RT: room temperature (about 25 ℃); w: week.
FIG. 9 comparison of ELP-DuIFN gamma activity changes at different storage temperatures; wherein, RT: room temperature (about 25 ℃); w: week.
Detailed Description
Example 1
1. Materials and methods
1.1 Primary reagents
Restriction enzymes XhoI and SacI, T4 DNA ligase, 1kb DNA Marker from Fermentas; the plasmid small quantity extraction Kit, the gel recovery Kit and the Viral RNA/DNAextraction Kit are purchased from TaKaRa company; the prestained Protein molecular weight Marker is purchased from Biotechnology engineering (Shanghai) GmbH; DMEM medium, FM medium, pancreatin, fetal bovine serum were purchased from Thermo Fisher Scientific; MTS detection kit, BCA protein quantitative detection kit, SDS PAGE gel electrophoresis kit and Tris-Gly electrophoresis buffer are purchased from kang, century limited company.
1.2 Main biological Material and test animals
DH5 alpha, BL21(DE3) E.coli competent cells were purchased from Hakka century Co; DuIFN γ was synthesized by bio-engineering (shanghai) gmbh; pET30a-ELP vector and VSV (vesicular stomatitis virus) were offered by professor Sun Huai Chang, university of Yangzhou, and the construction of pET30a-ELP vector was disclosed in the open literature (Bank i et al, 2005), and pET30a-ELP vector is readily available to those skilled in the art based on the disclosure in the prior art; duck fibroblast (DEF) and canine kidney cell (MDCK) are preserved in a veterinary high-tech research focus laboratory of Jiangsu province; DHAV-1 (strain SH) was a gift from Shanghai veterinary research institute of Chinese academy of agricultural sciences; male BALB/c mice at 4 weeks of age were purchased from the university of Yangzhou comparative medicine center; a non-immune cherry valley duck of 1 day old was purchased from a duck farm in Taizhou.
1.3 design Synthesis of DuIFN gamma Gene sequences
Due to the relative conservation of duck IFN sequence, in the design of DuIFN gamma gene sequence, according to the preference of Escherichia coli codon, the sequence optimization is carried out by referring to DuIFN gamma (DQ855272) published by GenBank (shown as SEQ ID NO: 1), enzyme cutting sites SacI and XhoI are introduced at two ends, and the target gene is synthesized by biological engineering (Shanghai) GmbH and is connected with pUC57 vector and named as pUC57-DuIFN gamma.
1.4 primer design Synthesis and construction of recombinant expression plasmid
Analysis of enzyme cutting site of pET30a-ELP vector sequence, 1 pair of primers TAGAGCTCCGGCGCGCCGTGCTCTGGTTCTGCTCTGTT(Sac I)/CACTCGAGTTAGCAACGGCAACGTTTCGGAG(Xho I) was designed, and the primers were synthesized by Biotechnology engineering (Beijing) GmbH. The method comprises the following steps of carrying out PCR amplification on DuIFN gamma by taking pUC57-DuIFN gamma as a template, carrying out double enzyme digestion on an amplification product by Sac I and Xho I, connecting the amplification product with a pET30a-ELP vector at 16 ℃ overnight by utilizing T4 ligase, transforming the ligation product into an escherichia coli DH5 alpha competent cell by adopting a heat shock method, coating the escherichia coli DH5 alpha competent cell on an LB solid plate for culture at 37 ℃, then selecting a single colony with a regular shape for overnight culture, extracting a plasmid according to the operation steps of a plasmid extraction instruction, carrying out enzyme digestion by SacI/XhoI in a thermostatic water bath at 37 ℃, identifying a correct positive clone by 1.0% agarose gel, carrying out sequencing identification by a biological engineering (Beijing) stock Limited company, and identifying the correct recombinant plasmid named as pET30a-ELP-DuIFN gamma.
ELP and DuIFN γ different fusion sequences: in constructing the recombinant sequence, the choice of the fusion position of ELP and N-terminal (amino-terminal) or C-terminal (carboxyl-terminal) of the target protein depends mainly on the expression level of the target protein itself. The research preliminary experiment finds the expression quantity of ELP fused with amino terminal and carboxyl terminal of recombinant duck interferon respectively, thereby determining the sequence of the optimal construction sequence. pET30a-DuIFN gamma-ELP is constructed by the following steps: the DuIFN gamma gene is synthesized according to the preference optimization sequence of the codon of the escherichia coli, NdeI and BamHI are introduced into two ends (the specific sequence is the same as that of SEQ ID NO: 1 except enzyme cutting sites at the two ends), and the synthesized DuIFN gamma is subjected to double enzyme cutting by NdeI and BamHI and then is connected with pET30a-ELP vector which is subjected to double enzyme cutting by NdeI and BamHI to be screened to obtain pET30a-DuIFN gamma-ELP.
pET30a-DuIFN gamma recombinant vector without inserted ELP was used as a control: the pET30a-DuIFN gamma recombinant vector is constructed, and the Escherichia coli codon preference is synthesized by the company of Biotechnology engineering (Shanghai): 1 and introduces BamHI and NdeI at both ends. The synthesized DuIFN γ was double-digested with BamHI and NdeI, and then ligated with pET30a vector, which was also double-digested with BamHI and NdeI. After overnight at 16 ℃, the ligation products were transferred to DH5 α competent cells by heat shock and screened for recombinant clones. The recombinant clone identified correctly by BamHI and NdeI double enzyme digestion was sent to the company Limited of engineering bioengineering (Shanghai) for sequence determination to obtain pET30a-DuIFN gamma.
pET30a-ELP-DuIFN alpha recombinant vector was used as a control: with reference to the published duck interferon-alpha gene sequence (accession number: DQ861429) in GenBank, the genetic biological engineering (shanghai) incorporated by ltd, based on the codon preference of escherichia coli, synthesizes a gene sequence as shown in SEQ ID NO: 2 and introduced with XhoI and SacI at both ends. The synthesized DuIFN α was double-digested with XhoI and SacI, and then ligated with pET30a-ELP vector, which was also double-digested with XhoI and SacI. After overnight at 16 ℃, the ligation products were transferred to DH5 α competent cells by heat shock and screened for recombinant clones. The recombinant clone identified correctly by XhoI and SacI double enzyme digestion is sent to the company Limited of the engineering bioengineering (Shanghai) for sequence determination, and pET30a-ELP-DuIFN alpha is obtained.
1.5 recombinant interferon expression and purification
Reference methods for inducible expression of pET30a-ELP-DuIFN γ, pET30a-DuIFN γ -ELP, pET30a-DuIFN γ, pET30a-ELP-DuIFN α and pET30a-ELP and purification of recombinant proteins[17]Finally, protein purity was analyzed by 10% SDS-PAGE gel. After the protein was measured by BCA method, it was sterilized by filtration through a 0.22 μm filter and stored at-80 ℃ until use.
1.6 evaluation of safety of recombinant Interferon
1.6.1 cytotoxicity assays
MDCK and DEF were inoculated into 96-well plates, and when the cells grew well, 100. mu.L of recombinant protein ELP-DuIFN γ (n. 6) diluted in DMEM and FM medium in 10-fold gradient was added to each well, and the mixture was incubated at 37 ℃ with 5% CO2The cell culture box is used for culturing and recording the growth change condition of the cells. Meanwhile, cells without added protein are used as a control group. After 72h, the OD of each well is detected by using an MTS method490nmValue (n ═ 3), reference method[19]The percent growth inhibition (control OD) was determined490nmValue-proteomic OD490nmvalue)/OD of control group490nmThe value is multiplied by 100%, and the influence of ELP-DuIFN gamma on the growth of MDCK and DEF is judged.
1.6.2 Duck toxicity test
Methods described in the references [20]45 non-immunized cherry valley ducks aged 4 days were randomly divided into a blank control group, a sufficient dose interferon group (160. mu.g/kg) and a 10-fold diluted interferon group, each group consisting of 15 ducks. Interferon was administered by drenching, and the placebo group was fed normally. And observing and recording the feed intake, the excrement form and the mental state of the duck, and simultaneously measuring the body temperature.
1.7 half-life assay of recombinant Interferon
Reference reported cytopathic test method[13]And determining the in vivo and in vitro half-life period of the recombinant interferon ELP-DuIFN gamma. That is, 50% BALB/c mouse or cherry valley duck plasma is mixed with ELP-DuIFN gamma with final concentration of 10 mug/mL, and after water bath at 37 ℃ for 0h, 0.25h, 0.5h, 1h, 3h, 6h, 12h, 24h and 48h, antiviral activity (n is 3) of interferon in plasma at each time interval is detected according to cell growth conditions in MDCK-VSV system to judge in vitro half-life; or detecting the antiviral activity of the plasma of 0h, 6h, 12h, 24h, 36h, 48h, 72h and 96h after the non-immune cherry valley duck is administrated with the ELP-DuIFN gamma in the MDCK-VSV system to judge the half-life period in vivo. Plasma without ELP-DuIFN gamma was used as a blank.
1.8 detection of recombinant interfering in vitro antiviral Activity
1.8.1 VSV TCID50And DHAV-1TCID50、ELD50Measurement of
Reference to TCID related to Chinese veterinary dictionary50Requirement for assay method[21]MDCK cells and DEF cells were seeded in 96-well plates, and after the cells grew into monolayers, 100 μ L of VSV (n ═ 6) diluted in DMEM and FM medium at a 10-fold gradient was added to each well. Placing at 37 ℃ and 5% CO2After the cell culture box is cultured for 2h, the cell culture solution is discarded, 100 mu L of cell maintenance solution containing 2% FBS is inoculated into each hole for continuous culture, and cells without virus are set as negative control. Recording of cellular lesionsIn this case, until the cell state no longer changed, the TCID of VSV was calculated according to the Read-Muench method50. Determination of TCID of DHAV-1 in DEF cells in the same manner50ELD on Duck embryo50The value is obtained.
1.8.2 antiviral Activity assays
After MDCK and DEF cells were grown into monolayers, 10-fold dilutions of ELP-DuIFN γ (n. 6) were added to each well at 37 deg.C and 5% CO2Under the condition, after incubating for 12h, the culture solution is discarded, and then 100TCID is inoculated50The VSV of (1); a protein control group (only adding ELP-DuIFN gamma), a virus control group (only adding VSV) and a cell control group (not adding ELP-DuIFN gamma and not adding VSV) are arranged at the same time. After 24h, the antiviral activity of ELP-DuIFN γ (n-3) was calculated according to the Read-Muench method using the highest dilution of the virus control group in which 75% or more of the cells appeared diseased and the test group in which 50% or more of the cells did not appear diseased as one activity unit (U). The antiviral activity of ELP-DuIFN γ on DHAV-1 in DEF cells was determined in the same manner (n-3).
1.8.3 embryo body protection test
Detecting antiviral activity of ELP-DuIFN gamma on duck embryo, dividing 20 9-day-old duck embryos into 4 groups (5 per group), injecting physiological saline into allantoic cavity according to 100 μ L/one duck embryo of negative control group, and injecting 100ELD into allantoic cavity according to 100 μ L/one duck embryo of positive control group50DHAV-1 of (1), the protein control group was injected with 2.0mg/mL ELP-DuIFN γ into the allantoic cavity at a dose of 100. mu.L/mouse, and the antiviral test group was inoculated with 100ELD in the allantoic cavity of a duck embryo50DHAV-1 (100. mu.L/mouse) and 100. mu.L ELP-DuIFN γ. The death time and the number of the embryos of each group are observed and counted, and the antiviral effect of the embryos is analyzed.
1.9 detection of antiviral Activity in recombinant interfering Agents
1.9.1 LD of DHAV-150Measurement of
Reference to the Chinese veterinary dictionary related LD50Requirement for assay method[21]Dividing 40 4-day-old cherry valley ducks into 4 groups, and respectively administrating DHAV-1 stock solution and 10 μ L/duck in a dose of 100 μ L-1Diluent, 10-2Diluent, 10-3Diluting, observing and recording the feed intake, mental state, body temperature, feces, etc. of duck before and after challenge, recording the amount and time of DHAV-1 typical disease symptoms (mental depression, feces softness, lethargy, convulsion, and crossbow of body), and calculating LD of DHAV-150。
1.9.2 duckling protection test
35 cherry valley ducks aged 4 days were equally divided into 7 groups and tested in the manner of table 1: wherein the infectious dose of DHAV-1 is 100LD50The dose of ELP-DuIFN γ was given at 160. mu.g/kg every 2 d/time for 3 consecutive times. And observing the feed intake, the excrement form and the mental state of each group of ducklings, measuring the body temperature, and recording the number and time of ducks died of diseases in each group. Collecting the liver of the duck dying of illness and observing the pathological change condition.
TABLE 1 grouping of in vivo antiviral assays
1.10 detection of immunomodulatory effects of recombinant interferons
15 BALB/c mice (about 25 g-30 g) were divided into 3 groups on average, and ELP-DuIFN γ and ELP-DuIFN α were administered by drenching 2 times at a dose of 1 μ g/g, respectively, and the group was designated as a negative control group. Anticoagulated blood was collected from each mouse at 0, 4, 8, and 12d, and peripheral blood T lymphocytes were isolated. Th (helper T cell population CD 4) in peripheral blood T lymphocytes of different time periods of each mouse were analyzed and calculated by flow cytometry bicolor analysis+) And Tc (suppressor T cell population CD8+) Calculating the percentage of CD4+/CD8+Determining the immunoregulation function of ELP-DuIFN gamma.
1.11 recombinant Interferon stability assay
The purified ELP-DuIFN γ was divided into 4 portions, stored at Room Temperature (RT), 4 ℃, -20 ℃ and-80 ℃ respectively, and divided into several aliquots to avoid repeated freeze-thawing. Each group was assayed for interferon content weekly by direct SDS-PAGE, BCA, and for antiviral activity in MDCK-VSV system, and the stability of ELP-DuIFN γ at different storage temperatures was plotted by GraphPad Prism software.
2. Results
2.1 identification of recombinant expression plasmids
The restriction and identification results of the recombinant expression plasmid pET30a-ELP-DuIFN gamma show that the restriction generates about 7000bp and 450bp of target bands, which is consistent with the expectation (FIG. 1). Further sequencing results showed: the cloned DuIFN γ gene sequence has 100% homology with the optimized sequence. Indicating that the recombinant plasmid is correctly constructed.
2.2 expression and purification results of recombinant ELP-DuIFN. gamma. protein and ELP-DuIFN. alpha. protein
pET30a-ELP-DuIFN gamma-BL 21 was purified after induction expression, and SDS-PAGE analysis of protein expression and purification. The results show that the band of interest, mainly expressed in soluble form, appeared in conformity with the expected size, whereas the empty vector pET30-ELP control did not have the band of interest in the corresponding position (FIG. 2). The preliminarily purified recombinant protein is not dissolved in PBS, and the purity of the recombinant protein treated by the inclusion body purification reagent reaches more than 90 percent. The concentration of ELP-DuIFN γ measured by BCA method was 2.4. mu.g/. mu.L. The functional advantage of ELP is utilized to obtain the high-purity and high-concentration recombinant ELP-DuIFN gamma.
In addition, the research preliminary experiment searches expression quantities of ELP fused with the amino terminal and the carboxyl terminal of the recombinant duck interferon respectively, so as to determine the sequence of the optimal construction sequence. The results show that: the recombinant ELP-DuIFN gamma has high expression level and most of the recombinant ELP-DuIFN gamma can be expressed in a soluble way, and a large amount of target protein can be obtained by the ITC technology. Therefore, the ELP fused with the amino-terminal of the target protein (i.e., ELP-DuIFN gamma) was finally selected as the optimal sequence in this study. The expression result is presumably that ELP is fused with the amino terminal of the target protein due to the low self-expression level of duck interferon, and the ELP can be expressed in large amount in escherichia coli, so that the ELP is expressed firstly and then the target protein is expressed by utilizing the characteristic that the ELP is highly soluble protein, and the soluble expression of the foreign protein is promoted. The content of DuIFN gamma-ELP soluble target protein is less, and the content of ELP-DuIFN gamma soluble target protein is more. It is demonstrated that the fusion sequence of ELP and DuIFN gamma significantly affects the expression level of the soluble target protein.
The results with pET30a-DuIFN γ recombinant vector without ELP inserted as a control show that: (1) the pET30a-ELP-DuIFN gamma-BL 21 recombinant expression strain can express, the protein yield is about 240mg/L, the target protein size is about 81kDa, and the content of the target protein in a supernatant is higher, while the pET30a-ELP empty vector control has no band at the corresponding size; (2) the protein after induction of pET30a-DuIFN gamma-BL 21 is mainly inclusion body, the protein yield is about 100mg/L, the target protein is about 18.5kDa, and the empty vector control of pET30a has no band at the corresponding size. Indicating that ELP can promote the soluble expression of the recombinant interferon.
The ELP-DuIFN gamma fusion protein obtained by fusing ELP with DuIFN gamma can: (1) promoting soluble expression;
(2) simplified purification (salt ion is added, and purer protein can be obtained through centrifugation); (3) and the half-life period of the recombinant interferon is prolonged.
pET30a-ELP-DuIFN alpha-BL 21 was purified after induction of expression, and the recombinant protein ELP-DuIFN alpha purified by ITC method was detected by SDS PAGE method, and the results are shown in FIG. 3: a clear single-purpose band appears at 83ku, and the purity reaches over 90 percent. The BCA method determines that the concentration of ELP-DuIFN alpha is 2.2 mu g/mu L, the protein yield of each liter of bacterial liquid is about 220mg, and the recovery rate reaches 96%.
2.3 recombinant Interferon safety evaluation results
And (3) respectively acting gradient diluted ELP-DuIFN gamma on MDCK and DEF, detecting the activity of the cells by using an MTS method after 72 hours, and calculating the cell growth inhibition rate. The results are shown in Table 2: the growth inhibition rate of the cells by ELP-DuIFN gamma with different concentrations is 0, and the cells can normally proliferate. Meanwhile, the toxicity test result of the duckling also shows that: the ducklings of the ELP-DuIFN gamma group and the blank control group are fed normally, have excrement forming and good mental state, have body temperature in a normal range, and are safe and nontoxic with recombinant protein.
TABLE 2 growth inhibition of DEF and MDCK by ELP-DuIFN gamma
Table 2 Growth inhibition rates of ELP-DuIFNγon DEF and MDCK
Note: the initial concentration of ELP-DuIFN gamma is 2.4 mg/mL; x: an average; s: standard deviation of
2.4 half-Life of recombinant Interferon
The half-life of ELP-DuIFN gamma in BALB/c mice and cherry valley duck plasma was judged by the antiviral activity assay of interferon in MDCK-VSV system, and the results are shown in FIG. 4: the recombinant ELP-DuIFN gamma keeps stronger antiviral activity within 24 hours, the activity is still more than 50% until 48 hours, and the in vitro half-life period reaches 48 hours.
After the cherry valley duck is administrated with ELP-DuIFN gamma, the antiviral activity of interferon in anticoagulation blood collected at different time points is detected in an MDCK-VSV system, and the result is shown in a figure 5: negative plasma control has no antiviral activity, the in vivo antiviral activity of the cherry valley duck reaches 50% at about 18h after the administration of the recombinant ELP-DuIFN, then the activity continuously rises, reaches a peak at 36h, then the antiviral activity gradually decreases along with time extension, the activity is still higher than 50% at about 48h, and the antiviral activity does not exist at 96h, so the in vivo half-life period of the ELP-DuIFN gamma is more than 48 h.
2.5 recombinant Interferon in vitro antiviral Activity
TCID of VSV on MDCK cells according to the Read-Muench algorithm50Is 10-6.83TCID on DEF cells at 0.1mL50Is 10-6.170.1 mL; TCID of DHAV-1 on DEF cells50Is 10-5.360.1mL ELD on Duck embryos50Is 10-5.75/0.1mL。
The cell lesion inhibition method is used for detecting the antiviral activity of the recombinant ELP-DuIFN gamma in different cells, so that the cell growth of a cell control group and the cell growth of a protein control group are good, the cell lesion of a virus control group is obvious, and the cell state of an antiviral test group is obviously superior to that of the virus control group. The antiviral activity of ELP-DuIFN gamma in different systems is shown in Table 3 calculated by the Read-Muench method, the antiviral activity is shown in MDCK and DEF, and the activity in DEF-DHAV-1 is slightly lower than that in the other systems.
TABLE 3 ELP-DuIFN γ in vitro antiviral Activity assay results
Table 3 Antiviral activity of ELP-DuIFNγin different systems in vitro
The antiviral effect of the recombinant interferon was tested on SPF duck embryos and the statistical results are shown in Table 4: the positive control group died sequentially after 24h, and all died within 48 h; 2 duck embryos of the antiviral group die within 48h, and the negative control group, the protein control group and the antiviral group do not die after observation for 10d, which shows that the recombinant interferon can protect the duck embryos and has a strong antiviral effect.
TABLE 4 ELP-DuIFN gamma antiviral test results on Duck embryo
Table 4 Antiviral activity of ELP-DuIFNγon duck embryo
2.6 results of in vivo antiviral assay of recombinant interfering agents
According to the Read-Muench calculation rule, the LD of DHAV-1 in the duckling body50Is 10-3.3180.1 mL. Drenching 100LD according to each duckling50The test was carried out, and the statistical results after 10d are shown in Table 5: the positive group of ducklings begin to have clinical symptoms of mental depression, anorexia, head and neck backward tilting and the like within 48 hours, all ducklings die within 72 hours, and livers of ducks died of diseases have obvious bleeding points and necrotic foci; the blank control group fed normally has good mental state and growth state; the first-medicine-then-poison group and the first-medicine-then-poison group respectively have clinical symptoms and die at the beginning of 72 hours, and the protection rates of the first-medicine-then-poison group, the first-medicine-then-poison group and the medicine-poison group are respectively as follows: 40%, 60% and 60%.
TABLE 5 ELP-DuIFN γ in vivo antiviral assay results
2.7 optimal route of administration
The effect of different administration modes on the protective power of ELP-DuIFN gamma in the in vitro test is comparatively analyzed, and the result is shown in the table 6: the ducklings of the positive control group continuously have clinical symptoms of mental depression, anorexia, convulsion, crossbow shape of body and the like within 48h, and all die within 72 h; 3 ducks in the intramuscular injection administration group have clinical symptoms and die after 72 hours, and the protection rate is 40%; after 96 hours, the 2 ducks in the drenching administration group show clinical symptoms and die, and the protection rate is 60%; the blank control group did not die. The recombinant protein can delay the disease of the ducklings and even completely protect the ducklings from infection, and the drenching administration mode is superior to intramuscular administration.
TABLE 6 analysis of optimal route of administration
2.8 immunomodulation by recombinant interferons
The percentage of Th, Tc in the various groups of peripheral blood T lymphocytes at different time points was analyzed and determined by flow cytometry, see FIG. 6, and CD4 was calculated+/CD8+The ratio, results are shown in FIG. 7: when interferon is not administrated, the ratio of protein drenching group is equal to that of negative control group, and each group of CD4 in 4d+/CD8+The ratio is increased completely, the negative group is continuously and slowly increased, and becomes stable after 8 days; CD4 of ELP-DuIFN alpha group after 4d+/CD8+The value is small and the amplitude is stably increased; while ELP-DuIFN gamma group showed a large increase after 8 d. Shows that the recombinant ELP-DuIFN gamma can improve CD4 in mice+/CD8+The ratio can enhance the immunity of the organism, thereby enhancing the antiviral effect, and the immunoregulation capability of ELP-DuIFN gamma is better than that of ELP-DuIFN alpha.
2.7 recombinant Interferon stability
The results of visual observation by SDS-PAGE and quantification of the change in interferon content at different storage temperatures and different time points by BCA were shown in FIG. 8: the interferon stored at-80 ℃ is prolonged with time, the content of the recombinant interferon is not obviously changed (P is more than 0.05), and the stability is best when the interferon is stored at-80 ℃; the interferon stored at-20 deg.C and 4 deg.C is slowly reduced with time, and has basically the same change, and is lower than the group at-80 deg.C and higher than the group at room temperature; the interferon content stored at room temperature is prolonged along with the time, the reduction amplitude is large, and the interferon content of the recombinant duck stored at the temperature of minus 80 ℃ is reduced by about 30% when the recombinant duck is stored for 12 weeks.
The activity of ELP-DuIFN γ at different storage temperatures and different times was determined on a MDCK-VSV system and the results are shown in FIG. 9: ELP-DuIFN γ stored at-80 ℃ did not undergo significant changes in activity within 12 weeks (P > 0.05); ELP-DuIFN γ stored at room temperature decreased greatly over time, to about 65% at 12 weeks; ELP-DuIFN γ stored at-20 ℃ and 4 ℃ also decreased gradually over time, with the two groups at the same time having comparable activity.
3. Discussion of the related Art
Research shows that the carrier pET30a-ELP used in the experiment can obviously improve the yield of the soluble expression of the foreign protein and is also beneficial to efficiently purifying the foreign protein. However, since the soluble expression and purification recovery efficiency of the protein are affected by various factors, such as the amount of hydrophobic amino acids contained in the foreign protein, the expression level of the foreign protein itself, and the fusion sequence of the foreign protein and ELP[23,45]Therefore, duck interferon (DuIFN-. gamma.) was fusion expressed with the C-and N-termini of ELP in preliminary experiments. The results show that fusion of the C-terminus of ELP with DuIFN-gamma can better express soluble recombinant protein and facilitate purification by ITC method. The recombinant protein ELP-DuIFN gamma obtained by adopting an ELP C-terminal fusion mode in the research not only has simple purification, good purity (more than 90 percent), high recovery rate (96 percent), and the protein yield of each liter of bacterial liquid of 240mg, but also has the antiviral activity of 4.54 multiplied by 10 to the maximum extent4U/mg, fully embodies the functional advantages of ELP.
The research expresses DuIFN-gamma and ELP in a fusion mode, aims to overcome the defects that duck gamma-interferon has small molecular weight, is easy to degrade by protease and is discharged by kidney, has short half-life in vivo and cannot exert lasting curative effect by using ELP as an in vivo drug delivery carrier so as to achieve the purposes of stability and slow releaseThe purposes of releasing protein, prolonging the half-life period of ELP-DuIFN gamma, reducing the times of medication and reducing the application cost[25]. From research results, the half-life period of the recombinant DuIFN-gamma in the plasma of mice and ducklings is longer than that of the previously reported 12-24h[23,26]Approximately 48 hours, and administration of interferon every 48 hours is effective in protecting animals from viral challenge.
In the in vivo antiviral test, the study refers to Junzhen[27]The method compares the action effect of the recombinant protein in two different administration routes of intramuscular injection and drench administration, and also compares the influence of three administration modes of first administration and then toxicity, first administration and then toxicity and simultaneous toxicity on the activity of the recombinant protein, and the result shows that: the recombinant interferon and the virus are simultaneously drenched or firstly poisoned and then used, so that the antiviral effect is better, but the stress of animals can be reduced by simultaneously administering, and the method is the best administration mode. Meanwhile, the administration mode also meets the basic requirement of clinical convenience and rapidness, and is beneficial to the further development and application of the recombinant protein.
The viruses causing duck diseases are numerous, but most of the viruses do not cause diseases independently and can cause infected ducks to show clinical symptoms only under specific conditions[17]. The duck hepatitis virus type 1 (DHAV-1) is a virus which can cause diseases of ducklings to die independently, and mainly attacks ducklings within 4 weeks of age, particularly ducklings less than 1 week of age, and is most susceptible to infection, and the death rate is up to 90 percent[2]Is a representative virus causing waterfowl epidemic disease. DHAV-1 is selected as a research object in the research to evaluate the antiviral effect of the recombinant ELP-DuIFN gamma, and the research is also representative. The test result shows that: the recombinant ELP-DuIFN gamma can show higher antiviral activity on cells; in vivo, the disease time can be delayed, the duckling can be protected from being infected by DHAV-1, and the protection rate can reach 80%; and the results of embryo regression test and multiplex PCR detection further prove the high-efficiency inhibition effect of the recombinant ELP-DuIFN gamma on the virus. The research result lays a foundation for research and development of recombinant duck interferon preparations, prevention of other duck diseases and reduction of economic loss of duck breeding industry.
SEQ ID NO: 1 (reference to preferred codon optimized interferon gamma sequence):
TGCTCTGGTTCTGCTCTGTTCCTGGGTCAGCTGCAGAACGACATCGACAAACTGAAAGCTGACTTCAACGCTTCTAACTCTGACGTTGCTGACGGTAACCCGGTTTTCATCGAAAAAGTTAAAAACTGGACCGAACGTAACGAAAAACGTATCATCCTGTCTCAGATCGTTACCCTGTACCTGGAAATGCTGAAAAAAACCGACATGTCTAAACCGCACATCAAAAACCTGTCTGAACAGCTGAACACCCTGCGTAACACCCTGTCTAACGACTACAAAAAATTCCGTGACCTGGTTGAACTGTCTAACCTGCAGCTGACCGGTCTGAAAATCCAGCGTAAAGCTGTTTCTGAACTGTTCTCTGTTCTGCAGAAACTGGTTGAAACCTCTACCTCTAAACGTAAACGTTCTCAGTCTCCGAAACGTTGCCGTTGCGGCGCGCCG
SEQ ID NO: 2 (reference to preferred codon optimized interferon-alpha sequence):
TGCTCTCCGCTGCGTCTGCACGACTCTGCTTTCGCTTGGGACTCTCTGCAGCTGCTGCGTAACATGGCTCCGTCTCCGACCCAGCCGTGCCCGCAGCAGCACGCTCCGTGCTCTTTCCCGGACACCCTGCTGGACACCAACGACACCCAGCAGGCTGCTCACACCGCTCTGCACCTGCTGCAGCACCTGTTCGACACCCTGTCTTCTCCGTCTACCCCGGCTCACTGGCTGCACACCGCTCGTCACGACCTGCTGAACCAGCTGCAGCACCACATCCACCACCTGGAACGTTGCTTCCCGGCTGACGCTGCTCGTCTGCACCGTCGTGGTCCGCGTAACCTGCACCTGTCTATCAACAAATACTTCGGTTGCATCCAGCACTTCCTGCAGAACCACACCTACTCTCCGTGCGCTTGGGACCACGTTCGTCTGGAAGCTCACGCTTGCTTCCAGCGTATCCACCGTCTGACCCGTACCATGCGTGGCGCGCCG
reference to the literature
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[18]ZongYang,Xiao Tan,Jingjing Xiao,Xinyu Zhang,Xiaoli Xia,HuaichangSun,Half-life extension of porcine interferon-a by fusion to the IgG-binding domain of streptococcal G protein.Protein Expression and Purification 153(2019)53-58
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Sequence listing
<110> Jiangsu agriculture and animal husbandry science and technology occupational academy
Application of <120> duck interferon gamma fusion protein in preparation of medicine for preventing and treating duck viral hepatitis
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 444
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tgctctggtt ctgctctgtt cctgggtcag ctgcagaacg acatcgacaa actgaaagct 60
gacttcaacg cttctaactc tgacgttgct gacggtaacc cggttttcat cgaaaaagtt 120
aaaaactgga ccgaacgtaa cgaaaaacgt atcatcctgt ctcagatcgt taccctgtac 180
ctggaaatgc tgaaaaaaac cgacatgtct aaaccgcaca tcaaaaacct gtctgaacag 240
ctgaacaccc tgcgtaacac cctgtctaac gactacaaaa aattccgtga cctggttgaa 300
ctgtctaacc tgcagctgac cggtctgaaa atccagcgta aagctgtttc tgaactgttc 360
tctgttctgc agaaactggt tgaaacctct acctctaaac gtaaacgttc tcagtctccg 420
aaacgttgcc gttgcggcgc gccg 444
<210> 2
<211> 492
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tgctctccgc tgcgtctgca cgactctgct ttcgcttggg actctctgca gctgctgcgt 60
aacatggctc cgtctccgac ccagccgtgc ccgcagcagc acgctccgtg ctctttcccg 120
gacaccctgc tggacaccaa cgacacccag caggctgctc acaccgctct gcacctgctg 180
cagcacctgt tcgacaccct gtcttctccg tctaccccgg ctcactggct gcacaccgct 240
cgtcacgacc tgctgaacca gctgcagcac cacatccacc acctggaacg ttgcttcccg 300
gctgacgctg ctcgtctgca ccgtcgtggt ccgcgtaacc tgcacctgtc tatcaacaaa 360
tacttcggtt gcatccagca cttcctgcag aaccacacct actctccgtg cgcttgggac 420
cacgttcgtc tggaagctca cgcttgcttc cagcgtatcc accgtctgac ccgtaccatg 480
cgtggcgcgc cg 492
Claims (8)
1. A duck interferon gamma fusion protein ELP-DuIFN gamma is characterized in that the fusion protein is obtained by fusing an elastin-like polypeptide ELP and a protein amino terminal of duck interferon gamma.
2. The duck interferon gamma fusion protein ELP-DuIFN gamma of claim 1, wherein the preparation method of the fusion protein comprises: the synthesis of the amino acid sequence shown as SEQ ID NO: 1 and introducing SacI and XhoI enzyme cutting sites at two ends, inserting the DuIFN gamma gene between the SacI and XhoI enzyme cutting sites of pET30a-ELP vector, screening to obtain a recombinant vector pET30a-ELP-DuIFN gamma, converting the recombinant vector pET30a-ELP-DuIFN gamma into a competent expression strain to obtain an expression engineering bacterium, and performing induction expression, protein purification and endotoxin removal to obtain a fusion protein ELP-DuIFN gamma.
3. A codon optimized duck interferon gamma gene has a nucleotide sequence shown as SEQ ID NO. 1.
4. A duck interferon gamma fusion protein ELP-DuIFN gamma expression vector is characterized in that the construction method of the expression vector comprises the following steps: the synthesis of the amino acid sequence shown as SEQ ID NO: 1 and introducing SacI and XhoI enzyme cutting sites at two ends, inserting the DuIFN gamma gene between the SacI and XhoI enzyme cutting sites of pET30a-ELP vector, and screening to obtain recombinant vector pET30a-ELP-DuIFN gamma.
5. A transgenic cell line or an expression engineering bacterium containing the duck interferon gamma fusion protein ELP-DuIFN gamma expression vector of claim 4.
6. The use of the duck interferon gamma fusion protein ELP-DuIFN gamma of claim 1 in the preparation of a medicament for preventing and treating duck viral hepatitis.
7. Use of the duck interferon gamma fusion protein ELP-DuIFN gamma of claim 1 in the preparation of a medicament for resisting duck hepatitis 1 virus.
8. Use according to claim 6 or 7, wherein the medicament is administered by drench administration.
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| CN115028742A (en) * | 2022-06-21 | 2022-09-09 | 河北瑞兰生物科技有限公司 | Duck alpha interferon and duck interleukin 2 fusion protein and coding gene and application thereof |
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