CN114920817B - Pig interferon lambda 4 recombinant protein and preparation method and application thereof - Google Patents
Pig interferon lambda 4 recombinant protein and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and relates to a porcine interferon lambda 4 recombinant protein, a preparation method and application thereof. The amino acid sequence of the recombinant protein of the porcine interferon lambda 4 (sIFN-lambda 4) is shown as SEQ ID NO. 3. Ligating the sIFN-lambda 4 fragment to the plasmid to obtain a recombinant plasmid, wherein the nucleotide sequence of the sIFN-lambda 4 is shown as SEQ ID NO. 4; and (3) transforming the recombinant plasmid into escherichia coli, culturing a bacterial liquid, adding IPTG for induction, collecting inclusion bodies, dissolving and purifying to obtain the recombinant sIFN-lambda 4 protein. The recombinant sIFN-lambda 4 protein can induce gram negative bacteria such as escherichia coli, klebsiella pneumoniae, salmonella and the like to agglutinate so as to block invasion of eukaryotic cells, and can inhibit infection of porcine epidemic diarrhea virus at the cellular and animal body level. These results indicate that sIFN- λ4 has dual properties of blocking bacterial infection and antiviral infection.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a porcine interferon lambda 4 recombinant protein, a preparation method and application thereof.
Background
The presently discovered porcine enterocoronaviruses include porcine epidemic diarrhea virus (porcine epidemic diarrhea virus, PEDV), porcine transmissible gastroenteritis virus (transmissible gastroenteritis virus, TGEV), porcine delta coronavirus (porcine deltacoronavirus, PDCoV) and porcine enteroalpha coronavirus (porcine enteric alphacoronavirus, PEAV), mainly cause vomiting, diarrhea, dehydration and death of piglets, wherein the mortality rate of piglets up to 100% within 7 days of age causes serious economic loss to the pig industry. 4 porcine enterocoronaviruses have similar pathogenic mechanisms, the viruses enter the small intestine of a pig through oral-nasal infection, the coded S protein is combined with a receptor on the surface of villus epithelial cells of the small intestine to enter the cells, and the cells are greatly proliferated in the cells to cause the damage and dysfunction of the epithelial cells and villus atrophy, so that the nutrient malabsorption is caused, diarrhea and dehydration are further caused, and a great deal of death of infected piglets is finally caused. For pig intestinal coronavirus infection, prevention and control are mainly carried out through vaccine immunity, no specific therapeutic drug exists at present, and only commercial vaccines for preventing PEDV and TGEV infection exist at present, and safe and effective commercial vaccines for PEAV and PDCoV exist, so that development of effective therapeutic drugs for pig intestinal coronavirus infection is very important.
At present, antibiotics are mainly added into feed for preventing bacterial infection in the livestock and poultry breeding industry, but the long-term use of the antibiotics accelerates the variation of bacteria, evolves a plurality of different drug resistance mechanisms and leads to the generation of super drug resistance bacteria. When the intestinal mucosa barrier of live pigs is destroyed by viruses, the super bacteria can enter while the live pigs are still deficient, so that the virus infection is converted into virus-super bacteria mixed infection, and the effect of antibiotics or antiviral drugs is not obvious or even ineffective after the antibiotics or antiviral drugs are used.
Interferon (IFN) is a class of cytokines with broad-spectrum antiviral activity. IFNs are classified into three types, I, II, and III, depending on the receptor. The type I IFN (IFN-alpha/beta) and the type III IFN (IFN-lambda s) of mammals have strong antiviral activity, while the type II IFN (IFN-gamma) has weaker antiviral activity and mainly participates in the immune regulation in the process of converting natural immunity into acquired immunity. Recent studies have shown that the receptor for IFN-. Lambda.s (IFNLR) is highly expressed in epithelial cells of barrier organs such as intestinal tract. In contrast, the type I interferon receptor (IFNAR) is not expressed or expressed in very low abundance in intestinal epithelial cells. Thus, IFN- λs has a stronger advantage in treating enteroviral infectious diseases than type I interferon. The currently discovered pig IFN- λs has IFN- λ1, IFN- λ3 and IFN- λ4, and has more reports on the antiviral activity of the pig IFN- λ1 and IFN- λ3, and has very few reports on the preparation method and the function research of the pig IFN- λ4.
Disclosure of Invention
The invention aims at providing a recombinant protein of porcine interferon lambda 4 (sIFN-lambda 4), wherein the amino acid sequence of the recombinant protein of sIFN-lambda 4 is shown as SEQ ID NO. 3.
It is a second object of the present invention to provide a method for preparing sIFN-. Lambda.4 recombinant protein.
The invention also aims to provide the application of the sIFN-lambda 4 recombinant protein in preparing medicines for treating porcine epidemic diarrhea virus infection, other porcine enterocoronavirus infection and blocking gram negative bacteria infection.
The invention is realized by the following technical scheme:
prokaryotic expression of sIFN- λ4 recombinant protein
First, the amino acid sequence of sIFN-. Lambda.4 was optimized based on the gene sequence of porcine IFN-. Lambda.4 (sIFN-. Lambda.4) published on NCBI (GeneID: 102161030), specifically: the signal peptide of the sIFN- λ4 protein was removed and 6 histidines were introduced at the C-terminus of sIFN- λ4. The amino acid sequence of the optimized sIFN-lambda 4 recombinant protein is shown as SEQ ID NO. 3, and the nucleotide sequence of the coding sIFN-lambda 4 recombinant protein is shown as SEQ ID NO. 4. Designing a specific primer to amplify the optimized sIFN-lambda 4 gene, and connecting the obtained fragment to pET-15b (+) to obtain recombinant plasmid pET-15 b-sIFN-lambda 4. pET-15 b-sIFN-lambda 4 is transformed into escherichia coli BL21 (DE 3), and the high-efficiency expression of sIFN-lambda 4 in escherichia coli is proved by IPTG induction and SDS-PAGE detection, and the sIFN-lambda 4 exists mainly in an inclusion body form.
Large scale expression and purification of sIFN-lambda 4 recombinant proteins
And (3) a large amount of pET-15 b-sIFN-lambda 4 expression bacteria are induced, the bacteria are collected by centrifugation, and after the bacteria are crushed by a high-pressure crusher, the sediment is collected by centrifugation. The inclusion bodies were obtained by washing with washing solution (1% v/v Triton X-100,5mmol/L EDTA,20mmol/L Tris-HCl,0.1mol/L NaCl, pH 8.5) 2 times, centrifuging and collecting the pellet.
The inclusion bodies were dissolved in a solution (8 mol/L urea, 50mmol/L Tris-HCl,300mmol/L NaCl, pH 8.5), and the supernatant was collected by centrifugation and filtered through a 0.45 μm filter. The filtrate was purified using a Ni focus 6FF (IMAC) gravity column. Diluting with diluted solution (6 mol/L urea, 100mmol/L NaCl,50mmol/L Tris-HCl, pH 8.5), sequentially adding 1 # renaturation solution (4 mol/L urea, 0.5mol/L arginine, 2mmol/L reduced glutathione, 0.5mmol/L oxidized glutathione, 20mmol/L Tris-HCl, pH 8.5), 2 # renaturation solution (3 mol/L urea, 0.5mol/L L arginine, 2mmol/L reduced glutathione, 0.5mmol/L oxidized glutathione, 20mmol/L Tris-HCl, pH 8.5), 3 # renaturation solution (2 mol/L urea, 0.5mol/L L arginine, 2mmol/L reduced glutathione, 0.5mmol/L oxidized glutathione, 20mmol/L Tris-HCl, pH 8.5), 4 # renaturation solution (1 mol/L urea, 0.5mmol/L oxidized arginine, 2mmol/L Tris-HCl, pH8.5 mmol/L reduced glutathione, 2mmol/L Tris-HCl), 2mmol/L reduced glutathione (2 mmol/L Tris-HCl, pH8.5 mmol/L oxidized glutathione, pH8.5 mmol/L Tris-HCl), 2mmol/L oxidized glutathione, pH8.5 mmol/L Tris-HCl, pH 8.5. After the dialysis is completed, the protein solution is collected, and is concentrated to about 2mg/ml by ultrafiltration tube, and is stored below-70 ℃ after split charging. Meanwhile, the purity of the sample was detected by SDS-PAGE, and as a result, it was found that the purity of the obtained sIFN- λ4 was 95% or more.
Effect of sIFN-. Lambda.4 recombinant proteins on proliferation of PEDV in cells
IPI-FX cells (obtained from IPI-2I cell clones) cultured in 24-well cell culture plates were treated with different concentrations (1000 ng/ml, 1ng/ml and 0.001 ng/ml) of sIFN- λ4, respectively, and 24h later inoculated with the PEDV AJ1102 strain, while cell controls (neither treated with sIFN- λ4 recombinant protein nor vaccinated), blank controls (treated with 1000ng/ml of sIFN- λ4 alone, not vaccinated) and virus controls (positive control. Vaccinated but not treated with sIFN- λ4 recombinant protein) were established, and after cytopathy of the virus control wells, the virus was attenuated,after 3 freeze thawing times at-70 ℃, the TCID of each well sample was tested 50 . The results showed that sIFN-. Lamda.4 significantly inhibited the proliferation of PEDV AJ1102 strain on IPI-FX cells and was dose dependent.
Therapeutic Effect of sIFN- λ4 recombinant protein on piglets infected with PEDV
Healthy piglets which are negative for PEDV antigen antibodies of 5-day-old PEDV AJ1102 strain are subjected to oral infection by cytotoxin, and the first group (a continuous administration group) and the second group (a high-dose single administration group) of piglets are subjected to intramuscular injection of sIFN-lambda 4 recombinant protein through the neck at 18h after the challenge, and the third group is a non-challenge administration group. Observing and recording the mental state, feeding and defecation conditions of piglets every day after the poison attack, respectively collecting anal swabs before the poison attack, 1d, 3d, 5d, 7d, 9d and 11d after the poison attack, and detecting the poison discharge condition through fluorescent quantitative RT-PCR; and (3) timely performing a section inspection on dead piglets, and performing a section inspection on the rest piglets at 11d after the toxicity attack, so as to observe pathological changes of the piglets.
The result shows that vomiting, watery diarrhea and dehydration appear in piglets of the toxin-counteracting and non-administration group, and all the piglets die (5/5), the small intestine of the piglets is transparent, the wall of the small intestine is thinned, the small intestine is filled with watery liquid, and villi of the small intestine is shed; the continuous administration group (administration is carried out for 4 times, once a day, 50 mug/head/time) and the high-dose once administration group (200 mug/head) have diarrhea of only 1 piglet at 18 hours after toxin attack, but the normal state is quickly recovered, the other piglets are healthy and alive, the survival rate of the two groups of piglets is 100 percent (5/5), and obvious pathological changes are not seen in the section inspection. The toxin expelling detection finds that all piglets before toxin expelling are not detected, low-level toxin expelling is detected for all piglets in 1d after toxin expelling, the toxin expelling amount of the piglets which are not administered after toxin expelling in 3d after toxin expelling is obviously increased and is maintained until death, the toxin expelling amount of the piglets in the toxin expelling administration group is obviously less than that in the non-administration group, and the toxin expelling is not detected for 4 piglets in the continuous administration group which is 11d after toxin expelling. The results show that the sIFN-lambda 4 recombinant protein has good therapeutic effect on piglets infected by PEDV.
Agglutination effect of sIFN-lambda 4 recombinant protein on gram-negative bacteria
Use physiological saline to treat log phase colibacillus (E.coli) and Klebsiella pneumonia poleOD of bacteria (K.pnumoniae) and Salmonella suis (S.tyrphiminuium) 600nm Respectively adjusting the values to 0.5, taking 1.5ml of each bacterial liquid, adding sIFN-lambda 4 recombinant protein to a final concentration of 20 mug/ml, setting a blank control with physiological saline, standing at room temperature for 15min, and measuring the OD of each bacterial liquid 600nm Values. The result shows that the treatment of sIFN-lambda 4 recombinant protein can obviously reduce the OD of 3 bacterial liquids 600nm Values. Since all 3 bacteria treated were gram-negative, it was demonstrated that the sIFN-. Lamda.4 recombinant proteins may have strong agglutination activity against gram-negative bacteria.
Effect of sIFN- λ4 recombinant protein on invasion of Salmonella suis into RAW263.7 cells
10 μl of Salmonella suis bacteria liquid in logarithmic growth phase is taken, an equal volume of sIFN-lambda 4 recombinant protein (final concentration of 20 μg/ml) or LL-37 bactericidal peptide (final concentration of 40 μg/ml, positive control), physiological saline (blank control) and BSA (final concentration of 20 μg/ml negative control) are respectively added, after 1h, each treated bacteria liquid is respectively added into RAW263.7 cell monolayers cultured by 24-well cell culture plates, the inoculum is sucked and removed at 37 ℃ for 1h, and uninjured bacteria in the culture plates are washed out by PBS containing gentamycin. After lysing the cells with sterile cell lysates, the number of salmonella in the cells of each well was determined and the invasion rate was counted (invasion rate = total number of bacteria in the cell/total number of bacteria inoculated). The result shows that the sIFN-lambda 4 recombinant protein can obviously reduce the invasion rate of salmonella in pigs to RAW263.7 cells, which indicates that the sIFN-lambda 4 recombinant protein has stronger blocking capability to salmonella in pigs.
The beneficial effects of the invention are as follows:
when the sIFN-lambda 4 recombinant protein is prepared, the signal peptide of the sIFN-lambda 4 protein is removed, and 6 histidines are introduced into the C end of the sIFN-lambda 4, so that the escherichia coli can efficiently express the sIFN-lambda 4 recombinant protein, and the expression quantity reaches 100mg/L.
The sIFN-lambda 4 recombinant protein prepared by the invention has good treatment effect on piglets infected by PEDV, and the effect of a small-dose continuous administration group is better than that of a high-dose single administration group. Meanwhile, the sIFN-lambda 4 recombinant protein prepared by the invention has better agglutination effect on gram-negative bacteria and effect of blocking invasion of eukaryotic cells.
Drawings
Fig. 1: SDS-PAGE electrophoresis of expressed sIFN-lambda 4 recombinant protein. M: protein Marker;2: inducing the pET-15b (+) transformed bacteria for 6 hours; 3 to 6: inducing pET-15 b-sIFNlambda-4 transformant for 2, 4, 6 and 8 hours; 7: inducing pET-15b (+) transformed bacteria for 6 hours, crushing, and centrifuging the supernatant; 8: inducing pET-15b (+) transformed bacteria for 6 hours, crushing, and centrifuging to precipitate; 9: inducing pET-15 b-sIFN-lambda 4 transformant for 6 hours, crushing, and centrifuging the supernatant; 10: inducing pET-15 b-sIFN-lambda 4 transformant for 6 hours, crushing, and centrifuging to precipitate;
fig. 2: SDS-PAGE electrophoresis of recombinant protein for expression of purified sIFN- λ4. M: protein Marker;1: purified sIFN-lambda 4 recombinant protein;
fig. 3: for TCID 50 Detecting the inhibition effect diagram of sIFN-lambda 4 recombinant protein on PEDV proliferation;
fig. 4: the recombinant protein sIFN-lambda 4 is used for treating clinical manifestations and lesions of piglets infected with PEDV. A: continuously administering the piglets after toxin expelling; b: high-dose single administration group piglet after toxin expelling; c: the group piglets are not treated by detoxification; d: after toxin is removed, the piglet split inspection results of the group are continuously fed; e: the piglet split inspection result of the single high-dose administration group after toxin attack; f: the piglet split inspection results of the group without toxin attack;
fig. 5: detecting the agglutination effect of sIFN-lambda 4 recombinant proteins on different gram-negative bacteria by a cuvette method;
fig. 6: blocking effect of the recombinant sIFN-lambda 4 protein treatment on invasion of salmonella in pigs into RAW263.7 cells.
Detailed Description
The following detailed description of the present invention is provided to facilitate understanding of the technical solution of the present invention, but is not intended to limit the scope of the present invention.
PEDV AJ1102 strain (GenBank accession number: MK 584552) was isolated, identified and maintained by laboratory isolation of major laboratory viruses from agricultural microbiology national center of china university (jin Bi, songlin Zeng, shaobo Xiao, huanchun Chen, liurong fang. Complete genome sequence of porcine epidemic diarrhea virus strain AJ1102isolated from a suckling piglet with acute diarrhea in child. Journal of virology 2012,86 (19): 10910-10911).
Example 1: prokaryotic expression and purification of sIFN- λ4 recombinant protein:
1. construction of prokaryotic expression plasmid for expressing sIFN-lambda 4 recombinant protein
The amino acid sequence of sIFN- λ4 was optimized according to the gene sequence of porcine IFN- λ4 (sIFN- λ4) published on NCBI (GeneID: 102161030, the expressed amino acid sequence of which is shown in SEQ ID NO: 5), specifically: the signal peptide of the sIFN- λ4 protein was removed and 6 histidines were introduced at the C-terminus of sIFN- λ4. The amino acid sequence of the optimized sIFN-lambda 4 is shown as SEQ ID NO. 3, and the nucleotide sequence for encoding the sIFN-lambda 4 recombinant protein is shown as SEQ ID NO. 4. Primers lambda 4-P1 (shown as SEQ ID NO: 1) and lambda 4-P2 (shown as SEQ ID NO: 2) of the sIFN-lambda 4 gene after specific amplification optimization are designed. Culturing LLC-PK1 cells, inoculating Sendai virus when the cultured LLC-PK1 cells grow to a single layer, and collecting cells after 12h of virus inoculation. Extracting total RNA of cells as a template, and obtaining sIFN-lambda 4 genes with signal peptides removed by RT-PCR amplification by using primers lambda 4-P1 and lambda 4-P2, wherein 6 histidines are introduced into the C end of the sIFN-lambda 4. The obtained amplified product and prokaryotic expression vector pET-15b (+) are respectively subjected to double digestion by using NcoI and XhoI, and then are connected by using T4 DNA ligase to obtain recombinant plasmid pET-15 b-sIFN-lambda 4.
Expression and detection of sIFN- λ4 in E.coli
BL21 (DE 3) competent cells are respectively transformed by the constructed recombinant plasmid pET-15 b-sIFN-lambda 4 and the vector plasmid pET-15b (+) and coated with LB plates containing 100mg/L ampicillin, and the plates are cultured for 12 to 14 hours at a temperature of 37 ℃. Picking single colony in LB culture medium containing 100mg/L ampicillin, shaking culturing at 37deg.C 200r/min to bacterial liquid OD 600nm When the value reaches 0.4-0.6, adding isopropyl thio-beta-D-galactoside (IPTG) with the final concentration of 0.8mmol/L for induction expression. And respectively taking bacterial liquid converted from pET-15 b-sIFN-lambda 4 and bacterial liquid converted from pET-15b (+) after induction for 2h, 4h, 6h and 8h, centrifuging at 4 ℃ for 10min at 5000r/min, collecting bacterial bodies, and carrying out SDS-PAGE detection. Simultaneously taking bacterial liquid for inducing expression for 6 hours, centrifuging, then re-suspending the bacterial liquid by PBS, and carrying out ultrasonic treatment on the re-suspended bacterial liquidCrushing by a crusher, centrifuging at 4 ℃ for 10min at 5000r/min, and respectively taking supernatant and precipitate for SDS-PAGE detection.
The detection shows that the sIFN-lambda 4 recombinant protein can be efficiently expressed in the escherichia coli, reaches the expression peak after induction for 6 hours, and the expressed protein mainly exists in an inclusion body form, and the expression quantity is about 100mg/L, as shown in figure 1.
Large scale expression and purification of sIFN-lambda 4 recombinant proteins
3.1 Large scale expression of sIFN-lambda 4 recombinant proteins
20. Mu.l of bacterial liquid expressing sIFN-lambda.4 recombinant protein is added into 20ml of LB containing 100mg/L ampicillin, and after being mixed evenly, the bacterial liquid is cultured for 12 hours in a shaking table at 37 ℃ under 200 r/min. 10ml of the bacterial liquid was added to 1000ml of LB containing 100mg/L ampicillin. Mixing, shaking culture at 37deg.C with shaking table 200r/min to obtain bacterial liquid OD 600nm When the concentration is 0.4-0.6, adding IPTG with the final concentration of 0.8mmol/L for induction, collecting bacterial liquid after 6h of induction, centrifuging at 4 ℃ for 10min at 5000r/min, discarding the supernatant, resuspending bacterial precipitate with 100ml PBS, centrifuging at 4 ℃ for 10min at 5000r/min, and discarding the supernatant. The cells were resuspended in 100ml PBS, and after 5 times of disruption with a high-pressure disrupter at 800bar, the cells were centrifuged at 15000r/min at 4℃for 30min, the supernatant was discarded, and the pellet was collected. Washing with washing solution (1% v/v Triton X-100,5mmol/L EDTA,20mmol/L Tris-HCl,0.1mol/L NaCl, pH 8.5) for 2 times, centrifuging at 12000r/min at 4deg.C for 10min, discarding supernatant, and collecting precipitate to obtain inclusion body. To the collected inclusion bodies, 10ml of a solution (8 mol/L urea, 50mmol/L Tris-HCl,300mmol/L NaCl, pH 8.5) was added, and the inclusion bodies were dissolved by shaking in a shaker at 37℃for 2 hours at 160r/min, centrifuged at 15000r/min at 4℃for 30 minutes, and the supernatant was collected, mixed and filtered through a 0.45 μm filter.
3.2 Purification of sIFN- λ4 recombinant protein
Filling a gravity column with Ni Focus 6FF (IMAC) filler, balancing the gravity column with 2 column volumes of the solution, adding the inclusion body solution filtered in the step 3.1 into the column, and after passing through the column, washing with 3-4 times column volumes of the washing solution (500 mmol/L NaCl,20mmol/L imidazole, 50mmol/L NaH) 2 PO 4 pH 8.0) washing the column to wash out impurities, followed by eluting with eluent (100 mmol/L NaCl,250mmol/L imidazole, 20mmol/L Tris-HCl)pH 8.5), collecting the eluted protein, and diluting to 1mg/ml with eluent after measuring the concentration. Diluting the protein to 0.33mg/ml with a diluent (6 mol/L urea, 100mmol/L NaCl,50mmol/L Tris-HCl, pH 8.5), filling into dialysis bags, and sequentially dialyzing with a number 1 renaturation solution (4 mol/L urea, 0.5mol/L arginine, 2mmol/L reduced glutathione, 0.5mmol/L oxidized glutathione, 20mmol/L Tris-HCl, pH 8.5), a number 2 renaturation solution (3 mol/L urea, 0.5mol/L arginine, 2mmol/L reduced glutathione, 0.5mmol/L oxidized glutathione, 20mmol/L Tris-HCl, pH 8.5) for 8 hours, and further dialyzing with a number 3 renaturation solution (2 mol/L urea, 0.5mol/L L arginine, 2mmol/L reduced glutathione, 0.5mmol/L oxidized glutathione, 20mmol/L Tris-HCl, pH8.5 mmol/L reduced glutathione, 4 mmol/L oxidized glutathione, 0.5mmol/L reduced glutathione, pH 20.5 mmol/L arginine, pH8.5 mmol/L reduced glutathione, pH 8.5. After the completion of the dialysis with the renaturation solution No. 4, 1/2 of the dialysis solution was replaced with Tris-HCl buffer (100 mmol/L NaCl,50mmol/L Tris-HCl, pH 8.5) and dialyzed for 8 hours; repeating the above steps for 2 times; finally, the dialysis was performed with Tris-HCl buffer for 24 hours, during which the dialysis fluid was changed every 8 hours. And collecting protein solution after dialysis, ultrafiltering and concentrating to about 2mg/ml with ultrafiltration tube, packaging, and storing below-70deg.C. The purity of the recombinant sIFN- λ4 protein obtained by sampling and detecting the purity by SDS-PAGE is found to be more than 95%, as shown in figure 2.
Example 2: effect of sIFN- λ4 recombinant protein on proliferation of PEDV in cells
IPI-FX cells (obtained from IPI-2I cell clones) were seeded in 24-well cell culture plates, and after cell monolayer growth, sIFN-. Lamda.4 recombinant protein was added at concentrations of 1000ng/ml, 1ng/ml and 0.001ng/ml, 500. Mu.l per well, at 37℃with 5% CO 2 Acting for 24 hours in an incubator, and sucking and removing the added sIFN-lambda 4 recombinant protein; the PEDV AJ1102 strain was inoculated at an amount of 0.1MOI by washing 2 times with serum-free DMEM medium, 3 wells each, the inoculum was aspirated after 2 hours of inoculation, washed 2 times with serum-free DMEM medium, and serum-free DMEM medium containing 5 μg/ml pancreatin was added. Cell controls (neither treated with sIFN-. Lambda.4 recombinant protein nor toxic), blank controls (1000 ng +.ml of sIFN-. Lambda.4 recombinant protein treated, no virus challenge) and virus control (positive control. Toxin-receiving but not treated with sIFN- λ4 recombinant protein), 3 duplicate wells per control; containing 5% CO at 37 DEG C 2 Culturing in incubator (A), collecting virus after virus contrast hole cytopathy, freezing and thawing at-70deg.C for 3 times, and detecting TCID of each hole sample 50 。
The results showed that the sIFN-. Lamda.4 recombinant protein significantly inhibited the proliferation of the PEDV AJ1102 strain on IPI-FX cells and was dose-dependent, as shown in FIG. 3.
Example 3: therapeutic effect of sIFN-lambda 4 recombinant protein on piglets infected with PEDV
Piglets negative for both PEDV antigen and antibody at 15-day-old and 5-day-old are divided into 3 groups, 5 pigs are orally infected with 10 pigs each 6 TCID 50 The first group (continuous administration group) and the second group (high dose single administration group) of piglets were injected intramuscularly with sIFN-. Lamda.4 recombinant protein via the neck at 18h after challenge, wherein the first group was administered continuously 4 times a day at a dose of 50. Mu.g/head/time; the second group was injected once only at a dose of 200 μg/head; the third group is the group without toxin. Observing and recording the mental state, feeding and defecation conditions of piglets every day after the virus attack, respectively collecting anal swabs before the virus attack, 1d, 3d, 5d, 7d, 9d and 11d after the virus attack, and detecting the virus ejection condition by using primers PEDV-N-F (shown as SEQ ID NO: 6) and PEDV-N-R (shown as SEQ ID NO: 7) through fluorescent quantitative RT-PCR; and (3) timely performing a section inspection on dead piglets, and performing a section inspection on the rest piglets at 11d after the toxicity attack, so as to observe pathological changes of the piglets.
The results show that the diarrhea of the third group of 5 piglets starts from 18h to 24h after the poison is attacked, the ingestion is reduced, the symptoms of the piglets are gradually serious along with the time, vomiting, watery diarrhea and dehydration appear, the mental retardation is bad, the piglets die from the 5 th (2 heads), the 6 th (2 heads) and the 8 th (1 head) of the piglets after the poison is attacked, the diarrhea of the first group and the second group respectively appears at 18h after the poison is attacked, but the piglets return to normal quickly, the other piglets are healthy, and the survival rate of the piglets in the first group and the second group is 100% (5/5). The small intestine of the third group of piglets is transparent, the small intestine wall is thinned, the small intestine is filled with water-like liquid, and the small intestine villi fall off. No apparent lesions were seen in the small intestine of the first and second groups of piglets. As shown in fig. 4. The toxin expelling detection finds that all piglets before toxin expelling are not detected, all piglets in 1d after toxin expelling are detected to be low in toxin expelling level, the toxin expelling amount of the piglets in 3d and the third group after toxin expelling is obviously increased and is maintained until death, the toxin expelling amount of the piglets in the first group and the second group is obviously less than that of the piglets in the third group, and the toxin expelling amount of the piglets in the first group is not detected by the piglets in the 4 th group after toxin expelling after the 11 d.
The results show that the sIFN-lambda 4 recombinant protein has good treatment effect on piglets infected by PEDV, and the effect of a small-dose continuous administration group is better than that of a high-dose single administration group.
Table 1 fluorescent quantitative RT-PCR detection of piglet anal swab viral load
Remarks: "-" indicates no viral RNA was detected in the piglet anal swab and "×" indicates piglet death.
Example 4: agglutination activity of sIFN-lambda 4 recombinant proteins against three gram-negative bacteria
The overnight recovered escherichia coli, klebsiella pneumoniae and salmonella suis are transferred into fresh LB culture medium according to the volume ratio of 1:100, and are placed in a shaking table at 37 ℃ for 200r/min to be cultured to logarithmic phase. Centrifuging at 4000r/min for 10min, collecting thallus, washing twice with PBS, and adjusting bacterial liquid OD with sterile physiological saline 600nm The value was 0.5. Sucking 1.5ml of bacterial liquid into a 2ml centrifuge tube, adding sIFN-lambda 4 recombinant protein to a final concentration of 20 mug/ml, setting up a blank control (adding physiological saline with the same volume as sIFN-lambda 4), fully mixing uniformly, standing at room temperature for 15min, and measuring the OD of all bacterial liquids by a cuvette method 600nm Values.
The results show that OD of 3 gram-negative bacterial solutions after sIFN-lambda 4 recombinant protein is added 600nm The values are all obviously reduced, and the bacterial liquid OD added with physiological saline is added 600nm As shown in FIG. 5, the values did not change significantly, which indicates that the sIFN- λ4 recombinant protein can cause agglutination of Escherichia coli, klebsiella pneumoniae and Salmonella suis, all of which are gram-negative bacteria, and thus it is presumed that the sIFN- λ4 recombinant protein may have agglutination effects on all of the gram-negative bacteria.
Example 5: effect of sIFN- λ4 recombinant protein on invasion of Salmonella suis into RAW263.7 cells
The overnight resuscitated salmonella suis bacterial liquid is transferred into fresh LB culture medium according to the volume ratio of 1:100, and is placed in a shaking table at 37 ℃ for shaking culture at 200r/min to the logarithmic phase. Centrifuging at 4000r/min for 10min to collect thallus, suspending and washing twice with PBS, and adjusting bacterial liquid OD with sterile physiological saline 600nm Is 0.1 (bacterial concentration is about 1X 10) 8 CFU/ml). To 10. Mu.l of the bacterial liquid, an equal volume of physiological saline (Control) or sIFN-. Lamda.4 recombinant protein (final concentration 20. Mu.g/ml), BSA (final concentration 20. Mu.g/ml, negative Control) and LL-37 antibacterial peptide (final concentration 40. Mu.g/ml, positive Control) were added, and the mixture was thoroughly mixed and placed in an incubator at 37℃for 1 hour. Taking out the treated bacterial liquid, adding 980 μl of serum-free DMEM medium into each tube, shaking gently, adding RAW263.7 cells to grow into monolayer (cell amount 10) 5 Individual/well) in a 24-well cell culture plate. The inoculum was pipetted off at 37℃for 1h, the cells were washed 3-5 times with PBS containing gentamicin (200. Mu.g/ml), then 2 times with PBS, then 200. Mu.l of cell lysate containing 1% Triton X-100 was added, and the mixture was allowed to stand at room temperature for 10min after pipetting. Mu.l of the cell lysate was added to 450. Mu.l of sterile physiological saline, and after thorough mixing, serial 10-fold dilutions were performed with sterile physiological saline, each LB plate was applied with the bacterial solutions of different dilutions, and the colonies were counted after incubation at 37℃for 16 hours, and the invasion rate (invasion rate = total number of intracellular bacteria/total number of inoculated bacteria) was counted.
As a result, it was found that the bacterial invasion rate of the physiological saline and BSA treated group was about 10%, while the bacterial invasion rate of the sIFN-. Lamda.4 treated group was less than 0.01%, and the bacterial invasion rate of the LL-37 antimicrobial peptide treated group was about 0.0001%, as shown in FIG. 6, which revealed that the sIFN-. Lamda.4 recombinant protein was effective in blocking invasion of Salmonella in pigs into RAW263.7 cells.
The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention, so that all equivalent changes or modifications of the structure, characteristics and principles described in the claims should be included in the scope of the present invention.
SEQUENCE LISTING
<110> university of agriculture in China
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Claims (8)
1. The porcine interferon lambda 4 recombinant protein is characterized in that the amino acid sequence of the porcine interferon lambda 4 recombinant protein is shown as SEQ ID NO. 3.
2. A gene encoding the recombinant protein of porcine interferon lambda 4 as claimed in claim 1, wherein said gene is as shown in SEQ ID No. 4.
3. The method for preparing the recombinant protein of porcine interferon lambda 4 as claimed in claim 1, comprising the following steps:
step 1: connecting a gene for encoding the porcine interferon lambda 4 recombinant protein into a plasmid to obtain a recombinant plasmid, transforming the recombinant plasmid into escherichia coli competent cells, and culturing to obtain seed liquid; the nucleotide sequence of the gene for encoding the porcine interferon lambda 4 recombinant protein is shown as SEQ ID NO. 4;
step 2: performing expansion culture on the seed liquid, then adding IPTG to induce, and collecting bacterial liquid;
step 3: centrifuging, re-suspending, crushing under high pressure, centrifuging, and collecting precipitate to obtain inclusion body;
step 4: dissolving inclusion bodies, centrifugally collecting supernatant, and filtering to obtain inclusion body denaturation solution;
step 5: purifying, diluting, dialyzing and ultrafiltering the filtered inclusion body denatured solution to obtain the recombinant protein of the porcine interferon lambda 4.
4. The method for preparing a recombinant protein of porcine interferon lambda 4 according to claim 3,
in step 1, the plasmid was pET-15b (+), and the E.coli was BL21 (DE 3).
5. The method for preparing a recombinant protein of porcine interferon lambda 4 according to claim 3,
in the step 2, the E.coli bacterial liquid OD after the expansion culture 600nm Reaching 0.4-0.6, adding IPTG with final concentration of 0.8mmol/L and induction time of 6h.
6. Use of the recombinant protein of porcine interferon lambda 4 according to claim 1 or the gene according to claim 2 for the preparation of a medicament for the treatment of PEDV infection and for blocking gram negative bacterial infection, characterized in that the gram negative bacteria is escherichia coli, klebsiella pneumoniae or salmonella suis.
7. Use of the recombinant protein of porcine interferon lambda 4 according to claim 1 or the gene according to claim 2 for the preparation of a medicament for the treatment of PEDV infection.
8. Use of the recombinant protein of porcine interferon lambda 4 according to claim 1 or the gene according to claim 2 for the preparation of a medicament for blocking gram-negative bacterial infection, characterized in that the gram-negative bacteria is escherichia coli, klebsiella pneumoniae or salmonella suis.
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| CN104540849A (en) * | 2012-03-28 | 2015-04-22 | 美利坚合众国,由健康及人类服务部部长代表 | A novel interferon-lambda4 (ifnl4) protein, related nucleic acid molecules, and uses thereof |
| CN106222178A (en) * | 2016-08-08 | 2016-12-14 | 武汉大学 | A kind of recombinant interferon λ 4 coded cDNA sequence and its preparation method and application |
| CN110205323A (en) * | 2019-06-04 | 2019-09-06 | 军事科学院军事医学研究院军事兽医研究所 | Express the expressing gene, recombinant expression carrier, recombinant lactobacilli and its preparation method and application of 3 type interferon of pig |
| WO2021034101A1 (en) * | 2019-08-21 | 2021-02-25 | 한국과학기술원 | Novel interferon lambda variant and production method therefor |
| CN113425832A (en) * | 2020-03-23 | 2021-09-24 | 杭州先为达生物科技有限公司 | Use of interferon lambda in the treatment of infections with novel coronaviruses (2019-nCoV) |
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2022
- 2022-05-12 CN CN202210515788.4A patent/CN114920817B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104540849A (en) * | 2012-03-28 | 2015-04-22 | 美利坚合众国,由健康及人类服务部部长代表 | A novel interferon-lambda4 (ifnl4) protein, related nucleic acid molecules, and uses thereof |
| CN106222178A (en) * | 2016-08-08 | 2016-12-14 | 武汉大学 | A kind of recombinant interferon λ 4 coded cDNA sequence and its preparation method and application |
| CN110205323A (en) * | 2019-06-04 | 2019-09-06 | 军事科学院军事医学研究院军事兽医研究所 | Express the expressing gene, recombinant expression carrier, recombinant lactobacilli and its preparation method and application of 3 type interferon of pig |
| WO2021034101A1 (en) * | 2019-08-21 | 2021-02-25 | 한국과학기술원 | Novel interferon lambda variant and production method therefor |
| CN113425832A (en) * | 2020-03-23 | 2021-09-24 | 杭州先为达生物科技有限公司 | Use of interferon lambda in the treatment of infections with novel coronaviruses (2019-nCoV) |
Non-Patent Citations (1)
| Title |
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| "Type III Interferon Restriction by Porcine Epidemic Diarrhea Virus and the Role of Viral Protein nsp1 in IRF1 Signaling";Qingzhan Zhang 等;《Journal of Virology》;第92卷(第4期);doi.org/10.1128/jvi.01677-17 * |
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