CN113603766A - Duck interleukin-2 for immune enhancement and application thereof as immunopotentiator - Google Patents
Duck interleukin-2 for immune enhancement and application thereof as immunopotentiator Download PDFInfo
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- CN113603766A CN113603766A CN202110920822.1A CN202110920822A CN113603766A CN 113603766 A CN113603766 A CN 113603766A CN 202110920822 A CN202110920822 A CN 202110920822A CN 113603766 A CN113603766 A CN 113603766A
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Abstract
The invention discloses duck interleukin-2 for immune enhancement and application thereof as an immunopotentiator. The duck interleukin-2 protein is (a1) or (a 2): (a1) a protein consisting of amino acid residues shown in a sequence 1 in a sequence table; (a2) and (a1) derived protein which is obtained by substituting and/or deleting and/or adding (a1) by one or more amino acid residues and has the function of the immune-enhanced duck interleukin. The recombinant interleukin-2 product provided by the invention is a high-efficiency immunopotentiator, has a simple preparation method, can be applied to inactivated vaccines, live vaccines and subunit vaccines for poultry, and prolongs the immunity duration of organisms while improving the antibody titer of the organisms.
Description
Technical Field
The invention relates to duck interleukin-2 for immune enhancement and application thereof as an immunopotentiator.
Background
Interleukin-2 (IL-2) is one of the most important immunomodulatory factors with a wide range of biological activities, and in 1976 Morgan et al first discovered murine IL-2 in PHA-stimulated murine splenic lymphocyte culture supernatants, and was called T Cell Growth Factor (TCGF) because it promotes and maintains T cell growth. Formally named interleukin-2 in the second international lymphokine working conference held in switzerland in 5 months 1979. Which are secreted by specific antigens or mitogen-activated T-lymphocytes or T-lymphocyte cell lines to produce Th 1-type lymphokines. Can activate T cells, promote B cells to differentiate and secrete antibodies, induce and generate Interferon (IFN) and Tumor Necrosis Factor (TNF), enhance the killing activity of monocytes and natural killer cells (NK), improve the cellular immunity and humoral immunity of animal bodies, and have important regulation effect in the immune response of the bodies.
At present, poultry epidemic diseases are complex and various, vaccines are the first means for preventing and controlling the poultry diseases, inactivated vaccines and subunit vaccines commonly used in poultry vaccines at the present stage need a long time to excite an organism to generate immune response after being injected into the organism, at least 3-4 weeks are needed until the antibody titer reaches a certain level, and the vaccine has low antibody titer or is difficult to maintain a high level for a long time, so that the development of an efficient immune adjuvant is urgently needed, and the antibody titer and the immune duration of the vaccine are improved.
Disclosure of Invention
Therefore, the invention provides duck interleukin-2 for immune enhancement and application thereof as an immunopotentiator.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides duck interleukin-2 protein for enhancing immunity, wherein the duck interleukin-2 protein is (a1) or (a 2):
(a1) a protein consisting of amino acid residues shown in a sequence 1 in a sequence table;
(a2) and (a1) derived protein which is obtained by substituting and/or deleting and/or adding (a1) by one or more amino acid residues and has the function of the immune-enhanced duck interleukin.
Nucleic acid molecules encoding said proteins also belong to the scope of protection of the present invention.
Preferably, the nucleic acid molecule is a DNA molecule as shown in any one of (b1) to (b3) below:
(b1) the nucleotide sequence is a DNA molecule of a sequence 2 in a sequence table;
(b2) a DNA molecule having 75% or more identity to the nucleotide sequence defined in (b1) and encoding the protein of claim 1;
(b3) a molecule that hybridizes under stringent conditions to the nucleotide sequence defined in (b1) and encodes the protein of claim 1.
Expression cassettes, recombinant vectors, recombinant microorganisms or transgenic cell lines containing said nucleic acid molecules.
The recombinant microorganism is obtained by introducing the nucleic acid molecule into an escherichia coli strain BL21(DE3) to obtain recombinant escherichia coli BYSW 007.
The recombinant Escherichia coli BYSW007 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation number is CGMCC NO. 22853.
The recombinant Escherichia coli BYSW007 is applied to preparation of the protein.
The invention also provides application of the protein, which is at least one of the following (c1) - (c 8):
(c1) as an immunopotentiator;
(c2) preparing an immunopotentiator;
(c3) as an immunopotentiator for vaccines;
(c4) as an immunopotentiator for duck tembusu virus vaccines;
(c5) preparing a vaccine;
(c6) preparing a duck tembusu virus vaccine;
(c7) as an antiviral agent;
(c8) preparing the antiviral preparation.
Preferably, the immunopotentiator comprises the following components: each milliliter of the feed contains 10000IU of duck interleukin-2 protein, 1-5 mg of achyranthes polysaccharide and 50-200 mug of vitamin C; 5 ng-10 ng of levamisole, and the balance of phosphate buffer solution.
The invention also provides a method for preparing the duck interleukin-2 protein for immune enhancement, which comprises the following steps: and culturing the recombinant Escherichia coli BYSW007 to obtain the duck interleukin-2 protein.
The recombinant Escherichia coli BYSW007 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of Xilu No. 1 of Beijing Kogyo, Chaoyang, and the preservation number is CGMCC NO. 22853.
The invention has the following advantages:
the recombinant interleukin-2 product provided by the invention is a high-efficiency immunopotentiator, has a simple preparation method, can be applied to inactivated vaccines, subunit vaccines and live vaccines for poultry, and prolongs the immunity duration of organisms while improving the antibody titer of the organisms.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.
The structures, ratios, sizes, and the like shown in the present specification are only used for matching with the contents disclosed in the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions that the present invention can be implemented, so that the present invention has no technical significance, and any structural modifications, changes in the ratio relationship, or adjustments of the sizes, without affecting the effects and the achievable by the present invention, should still fall within the range that the technical contents disclosed in the present invention can cover.
FIG. 1 is a diagram showing the results of the double restriction enzyme digestion of the recombinant plasmid pET-D-IL-2 provided in the embodiment of the present invention, wherein M is DL5000 DNAmarker; 1, double enzyme digestion fragments of pET-D-IL-2;
FIG. 2 is a diagram showing the result of pCR identification of a recombinant plasmid according to an embodiment of the present invention, wherein M is DL1500DNA Marker; 1, pCR identification fragment;
FIG. 3 is a Western-blot analysis of duck interleukin-2 provided in the embodiments of the present invention, wherein M: protein Marker, 1: induction expression of bacteria, 2: negative control;
FIG. 4 is a graph of antibody blocking rate (PI%) of inactivated vaccine and subunit vaccine groups tested ducks when IL-2 provided by the embodiment of the invention is used as a vaccine component;
FIG. 5 is a graph of antibody blocking rate (PI%) of live vaccine test ducks when IL-2 provided by the embodiment of the invention is used as a vaccine component;
FIG. 6 is a graph of antibody blocking rate (PI%) of inactivated vaccine and subunit vaccine groups tested in conjunction with IL-2 as an immunopotentiator for the synergistic immunization of ducks provided by the examples of the present invention;
FIG. 7 is a graph of antibody blocking rate (PI%) of duck in live vaccine group when IL-2 provided in the present invention is used as an immunopotentiator for synergistic immunization;
FIG. 8 is a schematic view of loading a 96-well cell culture plate according to an embodiment of the present invention.
Detailed Description
The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the present invention, pET-30a (+) vector was purchased from Novagen, BL21(DE3) was purchased from kang, a century Biotechnology Co., Ltd.
Example 1 construction of Escherichia coli expressing Duck Interleukin-2
Artificially synthesizing an IL-2 gene by referring to a duck interleukin-2 gene sequence (Genbank accession number: AAO39413.1), wherein the sequence of the synthesized duck interleukin-2 (IL-2) is shown as a sequence 2 in a sequence table, and the sequence of an amino acid is shown as a sequence 1 in the sequence table, cloning the IL-2 gene into a prokaryotic expression vector pET30a (+), and transferring the prokaryotic expression vector into a prokaryotic expression strain BL21(DE3) to obtain a recombinant Escherichia coli BYSW007 capable of expressing the duck interleukin-2 gene, which is preserved in the common microorganism center of China Committee for Culture Collection of Microorganisms (CCMCC), and has the preservation number of CGMCC NO. 22853.
The construction process of the Escherichia coli for expressing the duck interleukin-2 comprises the following steps:
1. duck interleukin-2 gene synthesis: the IL-2 gene was synthesized by referring to the sequence of the duck interleukin-2 gene (Genbank accession No.: AAO39413.1), and adding Nde I cleavage site CATATG to the 5 'end and Xho I cleavage site CTCGAG to the 3' end.
The target gene primer sequence:
D-IL F 5'ATGTCTGACAAACCGGACATGGC 3'
D-IL R 5'TTATTTCAGCATAGAACGCAGG 3'
2. enzyme digestion of a target gene: the synthesized IL-2 gene was double-digested with restriction enzymes Nde I and Xho I, and the digestion system (20. mu.l): IL-2 Gene 1. mu.l, Nde I1. mu.l, Xho I1. mu.l, 10 XBuffer 2. mu.l, ddH2O15. mu.l, were worked up on ice, mixed and digested at 37 ℃ for 2 h. The digested fragments were separated by agarose gel electrophoresis and recovered and purified using a gel recovery kit.
3. And (3) carrying out enzyme digestion on the vector: the vector pET30a (+) was selected, and the vector pET30a (+) was double-digested with restriction enzymes Nde I and Xho I, and the digestion system (20. mu.l): pET30a (+) 1. mu.l, Nde I1. mu.l, Xho I1. mu.l, 10 XBuffer 2. mu.l, ddH2O15. mu.l, were worked up on ice, mixed and digested at 37 ℃ for 2 h.
4. And (3) connecting the target gene with the vector: the enzyme-cut product of the IL-2 gene and the enzyme-cut product of the vector pET30a (+) were ligated by using T4 ligase, and the ligation system (10. mu.l): 10. mu.l of Buffer, 2. mu.l of pET30a (+) cleavage product, 2. mu.l of interferon gene cleavage product, 1. mu.l of T4 DNA ligase, ddH2O4. mu.l, were worked up on ice, mixed and ligated overnight at 16 ℃.
5. And (3) transformation: and adding 10 mu l of the ligation product into centrifuge tubes containing 100 mu l of escherichia coli DH5 alpha competent cells, uniformly mixing, carrying out ice bath for 30min, transferring to a constant-temperature water bath heat shock at 42 ℃ for 90s, taking out, immediately carrying out ice bath for 2min, adding 500 mu l of LB liquid culture medium into each centrifuge tube, culturing at 37 ℃ and 200r/min for 1h, taking 100 mu l of the ligation product, coating a plate containing kanamycin, and carrying out inverted culture at 37 ℃ for overnight 14 h.
6. BYSW007 bacterial liquid culture and pET-D-IL-2 plasmid extraction: and (3) selecting a single colony on the plate, inoculating the single colony to an LB liquid culture medium containing kanamycin, culturing for 12h at 37 ℃ at 200r/min, harvesting a bacterial liquid of an escherichia coli strain BYSW007, and extracting plasmids according to the specification of a plasmid extraction kit.
7. Identification of pET-D-IL-2 recombinant plasmid
7.1 enzyme digestion identification: the recombinant plasmid was digested with Nde I and Xho I, and the digestion system (20. mu.l): pET-D-IL-2 recombinant plasmid 1. mu.l, Nde I1. mu.l, Xho I1. mu.l, 10 XBuffer 2. mu.l, ddH2O15. mu.l, were worked up on ice, mixed and digested at 37 ℃ for 2 h. The agarose electrophoresis revealed an approximately 5kb pET30a (+) vector band and a 510bp foreign fragment, which corresponded well with the expected size, and the results of the electrophoresis are shown in FIG. 1.
7.2PCR identification: the IL-2 target gene primer is adopted for PCR identification, the reaction system is 10 multiplied by PCR buffer 2 mul, 4 multiplied by dNTP (2.5mM)1 mul, the primers D-IL F and D-IL R (10 mul) are respectively 1 mul, the plasmid template is 0.1 mul, Taq enzyme (5U/mul) is 0.2 mul, and ddH is supplemented2O to a total volume of 20. mu.l. Reaction procedure 5min after pre-denaturation at 94 ℃; denaturation at 94 ℃ for 30 seconds, annealing at 52 ℃ for 30 seconds, extension at 72 ℃ for 30 seconds, and 30 cycles; extension at 72 ℃ for 7 minutes. The PCR product was detected by 1% agarose gel electrophoresis, a 510bp specific band was seen, the size was in full agreement with the expected size, and the electrophoresis results are shown in FIG. 2.
Example 2 purification of Duck Interleukin-2 protein by prokaryotic expression
Escherichia coli BYSW007 is transformed into competent cells of Escherichia coli BL21(DE3), and interleukin-2 protein is obtained through induced expression and purification. The biological activity of the protein is determined to be 1.26 multiplied by 105IU/ml; the protein content was 3.83 mg/ml.
Wherein, the prokaryotic expression and purification process of the duck interleukin-2 protein comprises the following steps:
1. fermentation culture: BL21(DE3) is transformed by pET-D-IL-2 plasmid, a single colony is picked up and inoculated into a proper amount of LB culture medium containing 50 ug/ml kanamycin, and shaking culture is carried out at 37 ℃ and 200r/min for overnight; inoculating the bacterial liquid into a sterilized fermentation tank according to 2% of the amount of the culture medium, ventilating and culturing at 37 ℃, controlling the stirring speed of the fermentation tank to be 500-700 r/min, controlling the dissolved oxygen to be 60-90% and controlling the pH value to be 7.0; when the thallus grows to the middle logarithmic growth stage, IPTG with the final concentration of 1mmol/L is added, and the induction is carried out for 5h at 37 ℃.
2. And (3) crushing thalli, centrifugally collecting thalli precipitates after the culture is finished, washing the thalli precipitates for 2 times by using PBS (phosphate buffer solution) to prepare 10% PBS suspension, crushing bacteria by using a high-pressure homogenizer at the temperature of 2-8 ℃, centrifuging the crushed bacteria liquid for 15 minutes at 8000r/min, and collecting inclusion bodies.
3. And (3) purifying the recombinant protein, washing the inclusion body by using a buffer solution containing 2M urea, washing for 30 minutes by using a magnetic stirrer, centrifuging for 10 minutes at 8000r/min at 4 ℃, removing a supernatant, and then repeatedly washing once. Adding 8mol/L urea to dissolve and wash the inclusion body, carrying out ultrasonic cleaning in an ice bath for 30 minutes, centrifuging at 8000r/min for 20 minutes to remove precipitates, taking supernatant, namely an inclusion body dissolving solution, carrying out overnight treatment at 4 ℃, carrying out renaturation on the solution on ultrafiltration renaturation equipment, changing the solution according to the volume of 5-10, after the renaturation is finished, centrifuging at 8000r/min to remove precipitates, collecting supernatant, adding the supernatant into a chromatographic column at the speed of 0.3 ml/min, carrying out linear gradient elution with a phosphate buffer solution with the pH of 7.0 at the speed of 1 ml/min, detecting with an ultraviolet detector at the wavelength of 280nm, collecting a target elution peak, and filtering and sterilizing with a filter membrane of 0.22 mu m. The protein content was determined to be 3.83mg/ml by BCA method.
4. Performing western-blot identification on the recombinant protein, performing SDS-PAGE electrophoresis on the recombinant protein, transferring the recombinant protein to a PVDF membrane at 100V for 70min, washing the membrane for 2 times by TBST, washing the membrane for 3 times by TBST after 1% BSA blocking solution is blocked at 37 ℃ for 1.5h, taking a rabbit-derived IL-2 monoclonal antibody as a primary antibody, adding 1:1000 diluent, incubating overnight at 4 ℃, washing the membrane for 3 times by TBST, adding 1:4000 diluted goat anti-rabbit IgG (HRP) secondary antibody, washing the membrane for 3 times by TBS, detecting by using a DAB (digital audio broadcasting) color development kit, and detecting the size of the recombinant protein in the induced recombinant thallus to be 18.7KDa which is consistent with the size of the IL-2 protein.
5. Determination of biological Activity of recombinant proteins under sterile conditions, recombinant proteins were diluted to 200IU per 1ml with assay medium and serially diluted 2-fold in 96-well cell culture plates for a total of 10 dilutions, each dilution being 2-well. Mu.l of the test solution was left in each well, and the excess solution in each well was discarded. CTLL-2 cells were run out of whole medium at 37 ℃ with 5% CO2Culturing under the condition of sufficient quantity, centrifuging to collect CTLL-2 cells, washing with RPMI 1640 culture solution 3 times, and then re-suspending in the basic culture solution to prepare each milliliterContaining 6.0X 105Cell suspension of individual cells at 37 ℃ with 5% CO2The cell suspension is added into 96-well cell culture plate containing standard solution and test solution 50 μ l per well, and the mixture is heated at 37 deg.C and 5% CO2Culturing for 18-24 hours under the condition, adding 20 mu l of MTT solution into each hole, and culturing at 37 ℃ and 5% CO2Culturing for 4-6 hr, adding 150 μ l of lysis solution into each well, and culturing at 37 deg.C with 5% CO2And preserving the heat for 18-24 hours under the condition, wherein the operations are carried out under the aseptic condition. And uniformly mixing the liquid in the cell plate, putting the cell plate into an enzyme-labeling instrument, measuring the absorbance at the position of 570nm of the wavelength by taking 630nm as a reference wavelength, and recording the measurement result. The test data is processed by computer program or four-parameter regression method to calculate protein biological activity of 1.26 × 105IU/ml.
Example 3 use of the Duck Interleukin-2 of the invention as an immunopotentiator or as a component of a vaccine
The duck interleukin-2 immunopotentiator provided by the embodiment of the invention comprises the following components: each milliliter of the feed contains 10000IU of duck interleukin-2 protein, 1-5 mg of achyranthes polysaccharide and 50-200 mug of vitamin C; 5 ng-10 ng of levamisole, the balance of phosphate buffer solution, adding a preservative benzyl alcohol according to the proportion of 0.1 percent of final concentration, and fully and uniformly stirring, wherein the concentration of the phosphate buffer solution is 0.01mol/L, and the pH value is 7.0.
The immunopotentiator can be used in combination with genetic engineering vaccines such as inactivated vaccines, live vaccines, subunit vaccines and the like. Can also be used as a vaccine component, and in the preparation process of genetic engineering vaccines such as conventional inactivated vaccines, live vaccines, subunit vaccines and the like, the duck interleukin-2 immunopotentiator is mixed into an antigen and then produced according to a vaccine process to obtain the immunogenicity enhanced vaccine. After the immunopotentiator is applied, the immunologic function of an animal body can be effectively enhanced; simultaneously, the immune efficacy of the conventional vaccine is effectively improved and the immune duration is prolonged.
Preparing duck tembusu inactivated vaccines A and B according to a conventional method, wherein A is a vaccine prepared by adding an immunopotentiator into a tembusu virus antigen; preparing duck Tembusu subunit vaccines C and D according to a conventional method, wherein C is TembusuVaccines in which an immunopotentiator is added to a subunit vaccine antigen; selecting 35-day-old healthy susceptible ducks (negative with DTMUV antigen and antibody) with 200 feathers, dividing the ducks into 5 groups, wherein each group contains 40 feathers, the 1 st group of immune vaccines A and the 2 nd group of immune vaccines B; group 3 immunization vaccine C, group 4 immunization vaccine D; group 5 was blank control group not immunized; carrying out secondary immunization 14 days after primary immunization according to the same mode, separately feeding the ducks of each group under the same conditions and environment, taking blood and separating serum before primary immunization, 7 days after primary immunization, 14 days after secondary immunization, 21 days, 1 month, 3 months, 4 months, 5 months and 6 months after secondary immunization, detecting the DTMUV ELISA antibody by feather, and counting and analyzing the change rule of the antibody. 10 DTMUV AX2015 strains are respectively selected from each group after blood collection for 14 days, 4 months, 5 months and 6 months after immunization for counteracting toxic substances, and 0.2ml (containing 10 AX2015 strains) is injected into each muscle4.0TCID50) And (5) collecting blood and separating serum 2 days after the virus counteracting, and carrying out DTMU virus separation.
Preparing duck tembusu attenuated live vaccines E and F according to a conventional method, wherein E is a vaccine obtained by adding an immunopotentiator into a tembusu virus antigen; selecting 35-day-old healthy susceptible ducks (negative with DTMUV antigen and antibody) 120 feathers, dividing into 3 groups, namely 40 feathers in each group, 6 th group of immune vaccine E and 7 th group of immune vaccine F; group 8 was a blank control group without immunization; blood is collected and serum is separated before and after 7 days, 14 days, 21 days, 1 month, 3 months, 4 months, 5 months and 6 months after the immunization, the DTMUV ELISA antibody is detected by feather-wise detection, and the change rule of the antibody is counted and analyzed. After blood sampling for 21 days, 4 months, 5 months and 6 months, 10 of the groups were treated with DTMUV AX2015 strain, 0.2ml (containing 104.0TCID50) of AX2015 strain was injected into each muscle of the feather, and blood was sampled and serum was separated 2 days after the treatment of the toxin, and DTMU virus isolation was performed.
According to the results of table 1 and fig. 4, when the immunopotentiator is applied to the preparation of genetic engineering vaccines such as inactivated vaccines, subunits and the like, the level of the DTMUV ELISA antibody can be remarkably improved, 7 days after first immunization, the mean blocking rates of the DTMUV ELISA antibody of the vaccine immunization groups A and C added with the immunopotentiator are 31.02% and 29.36% respectively, and the mean blocking rates of the DTMUV ELISA antibody of the vaccine immunization groups B and D added with no immunopotentiator are 8.57% and 0 respectively; then, the antibody water level gradually increases until 14 days after the second immunization, the mean blocking rates of the DTMUV ELISA antibodies of the vaccine immunization groups A and C added with the immunopotentiators are 81.16% and 80.37% respectively, and the mean blocking rates of the DTMUV ELISA antibodies of the vaccine immunization groups B and D added with no immunopotentiators are 59.88% and 55.64% respectively; until the antibody reaches the peak 21 days after the second immunization, the mean blocking rates of the DTMUV ELISA antibodies of the vaccine immunization groups A and C added with the immunopotentiators are 83.54 percent and 82.40 percent respectively, while the mean blocking rates of the DTMUV ELISA antibodies of the vaccine immunization groups B and D added with no immunopotentiators are 64.73 percent and 62.85 percent respectively; by 6 months after the second immunization, the mean blocking rates of the DTMUV ELISA antibodies of the vaccine immunization groups A and C added with the immunopotentiators are 49.37 percent and 46.53 percent respectively, while the mean blocking rates of the DTMUV ELISA antibodies of the vaccine immunization groups B and D added with no immunopotentiators are 21.35 percent and 12.01 percent respectively;
as can be seen from the results of table 2 and fig. 5, when the immunopotentiator is applied to the preparation of live vaccine, the DTMUV ELISA antibody level can be significantly increased, and 7 days after immunization, the DTMUV ELISA antibody blocking rate average value of the E vaccine immunization group to which the immunopotentiator is added is 30.04%, while the DTMUV ELISA antibody blocking rate average value of the F vaccine immunization group to which the immunopotentiator is not added is 5.88%; then the antibody water level gradually rises until the antibody reaches the peak at 21 days after immunization, the mean blocking rate of the DTMUV ELISA antibody of the E vaccine immunization group added with the immunopotentiator is 80.35%, and the mean blocking rate of the DTMUV ELISA antibody of the F vaccine immunization group without the immunopotentiator is 59.61%; by 6 months after the second immunization, the mean blocking rate of the DTMUV ELISA antibody of the E vaccine immunization group added with the immunopotentiator was 45.82%, and the mean blocking rate of the DTMUV ELISA antibody of the F vaccine immunization group without the immunopotentiator was 9.49%.
The serum antibody detection result shows that the DTMUV antibody blocking rate of the vaccine A, C, E group immunized ducks containing the immunopotentiator is far higher than that of B, D, F vaccine immunized ducks detected in the same period. The specific results are shown in tables 1 to 2 and FIGS. 4 to 5.
TABLE 1 inactivated vaccine and subunit vaccine test results of duck DTMUV ELISA antibody detection (blocking rate PI%)
Note: "-" indicates that the antibody blocking rate was negative.
TABLE 2 live vaccine test results of duck DTMUV ELISA antibody (blocking rate PI%)
Note: "-" indicates that the antibody blocking rate was negative.
TABLE 3 inactivated vaccine and subunit vaccine challenge test results 14 days, 4 months, 5 months, 6 months after immunization
TABLE 4 live vaccine challenge test results 14 days, 4 months, 5 months, 6 months after secondary immunization
The result of the challenge shows that, in the inactivated vaccine and subunit vaccine groups, the challenge is carried out 14 days after the second immunization, the DTMU virus separation after the challenge of the duck in the nonimmunized blank control group is 10/10 positive, the DTMU virus separation after the challenge of the A, C vaccine immunized group duck added with the immunopotentiator is 10/10 negative, the protection rate is 100%, the DTMU virus separation after the challenge of the duck in the B, D vaccine immunized group without the immunopotentiator is 2/10 positive, and the protection rate is 80%; 4 months after secondary immunization, the immunoprotection rates of A, C vaccines are 100%, and the immunoprotection rates of B, D group vaccines are 80% and 70% respectively; 5 months after secondary immunization, the immunoprotection rates of A, C vaccines are 100%, and the immunoprotection rates of B, D group vaccines are 10% and 0 respectively; 6 months after secondary immunization, the immune protection rates of the A, C vaccines are 80% and 60% respectively, and the immune protection rates of the B, D vaccines are 0;
the results of the challenge in table 4 show that the live vaccine group, the challenge 21 days after immunization, the DTMU virus separation after challenge of the nonimmunized blank control group ducks were 10/10 positive, the DTMU virus separation after challenge of the E vaccine immunization group ducks added with the immunopotentiator was 10/10 negative, the protection rates were 100%, while the DTMU virus separation rates after challenge of the E vaccine immunization group ducks not added with the immunopotentiator were 2/10, respectively, and the protection rates were 80%. 4 months after immunization, the immune protection rate of the E vaccine is 100 percent, and the immune protection rate of the F group vaccine is 70 percent; 5 months after immunization, the immune protection rate of the E vaccine is 100 percent, and the immune protection rate of the F group vaccine is 10 percent; at 6 months after immunization, the immune protection rate of the E vaccine was 80%, while the immune protection rate of the F vaccine was 0.
The challenge results at different time points show that the challenge protection rate of vaccine A, C containing the immunopotentiator and DTMU of duck in immune group E are far higher than that of B, D and DTMU of duck in immune group F detected in the same period, the vaccine A, C added with the immunopotentiator and the immune protection period of E are obviously prolonged until the protection rate is not lower than 80% at 6 months after immunization, while the immune protection rate is obviously reduced after 4 months after immunization without adding the immunopotentiator B, D and the vaccine F, and the vaccine basically has no protection force after 5 months and 6 months after immunization.
Example 5 immunopotentiator IL-2 synergistic immunopotency
Preparing duck tembusu attenuated inactivated vaccine B and duck tembusu subunit vaccine D according to a conventional method, selecting 35-day-old healthy susceptible ducks (DTMUV antigen and antibody negative) with 200 feathers, dividing the ducks into 5 groups, wherein each group contains 40 feathers, the 1 st group is an IL-2+ B synergistic immune group, and the 2 nd group is a B immune control group; the group 3 is IL-2+ D cooperative immunization group, the group 4 is D immunization control group, and the group 5 is blank control group without immunization; carrying out secondary immunization 14 days after primary immunization in the same way, separately feeding each group of ducks under the same conditions and environment, collecting blood and separating serum 14 days after secondary immunization, detecting DTMUV ELISA antibody by feather, counting and analyzing antibody changeAnd (5) regularity. Blood is collected before first immunization, 7 days after first immunization, before second immunization, 14 days, 21 days, 1 month, 3 months, 4 months, 5 months and 6 months after second immunization, serum is separated, the DTMUV ELISA antibody is detected by feather-by-feather, and the change rule of the antibody is counted and analyzed. 10 DTMUV AX2015 strains are respectively selected from each group after blood collection for 14 days, 4 months, 5 months and 6 months after immunization for counteracting toxic substances, and 0.2ml (containing 10 AX2015 strains) is injected into each muscle4.0TCID50) And (5) collecting blood and separating serum 2 days after the virus counteracting, and carrying out DTMU virus separation.
Preparing a duck Tembusu attenuated live vaccine F by a conventional method, selecting 35-day-old healthy susceptible ducks (DTMUV antigen and antibody negative) 120 feathers, dividing into 3 groups, wherein each group contains 40 feathers, the 6 th group contains IL-2+ F synergistic immunity group, and the 7 th group contains F immunity control group; group 8 was a blank control group without immunization; blood is collected and serum is separated before and after 7 days, 14 days, 21 days, 1 month, 3 months, 4 months, 5 months and 6 months after the immunization, the DTMUV ELISA antibody is detected by feather-wise detection, and the change rule of the antibody is counted and analyzed. After blood sampling for 21 days, 4 months, 5 months and 6 months, 10 DTMUV AX2015 strains are respectively selected for counteracting the toxin, and 0.2ml (containing 10) of AX2015 strains is injected into each muscle of feather4.0TCID50) And (5) collecting blood and separating serum 2 days after the virus counteracting, and carrying out DTMU virus separation.
According to the results of the table 5 and the figure 6, the IL-2 product can obviously improve the immune antibody level of gene engineering vaccines such as inactivated vaccines, subunits and the like, 7 days after first immunization, the blocking rate average values of the DTMUV ELISA antibodies of the IL-2 product synergetic B and D vaccine immune groups are 31.34% and 31.06% respectively, and the blocking rate average values of the DTMUV ELISA antibodies of the B and D vaccine immune control groups are 8.92% and 0 respectively; then the antibody water level gradually increases until 14 days after the second immunization, the IL-2 product cooperates with the DTMUV ELISA antibody blocking rate average values of the B and D vaccine immunization groups to be 82.07 percent and 81.53 percent respectively, and the DTMUV ELISA antibody blocking rate average values of the B and D vaccine immunization control groups to be 60.24 percent and 55.40 percent respectively; until the antibody reaches the peak 21 days after the second immunization, the mean blocking rate of the IL-2 product cooperating with the DTMUV ELISA antibody of the vaccine immunization group B and the vaccine immunization group D is 83.76 percent and 83.08 percent respectively, while the mean blocking rate of the DTMUV ELISA antibody of the vaccine immunization control group B and the vaccine immunization control group D is 65.10 percent and 62.37 percent respectively; by 6 months after the second immunization, the IL-2 product cooperated with the mean blocking rates of the DTMUV ELISA antibodies of the B and D vaccine immunization groups were 48.44% and 47.61%, respectively, while the mean blocking rates of the DTMUV ELISA antibodies of the B and D vaccine immunization control groups were 20.18% and 10.53%, respectively.
According to the results of the table 6 and the figure 7, the IL-2 product can obviously improve the immune antibody level of the live vaccine, and 7 days after immunization, the average blocking rate of the IL-2 product and the DTMUV ELISA antibody of the F vaccine immune group is 31.00 percent, while the average blocking rate of the DTMUV ELISA antibody of the F vaccine immune control group is 6.32 percent; then the antibody water level gradually rises until the antibody reaches the peak 21 days after immunization, the average blocking rate of the IL-2 product and the DTMUV ELISA antibody of the F vaccine immunization group is 80.25 percent, and the average blocking rate of the DTMUV ELISA antibody of the F vaccine immunization control group is 58.52 percent; by 6 months after the second immunization, the mean blocking rate of the DTMUV ELISA antibody of the IL-2 preparation and F vaccine immunization group is 46.52%, while the mean blocking rate of the DTMUV ELISA antibody of the F vaccine immunization control group is 10.01%.
The serum antibody detection result shows that the DTMUV antibody blocking rate of the duck in the IL-2 product synergetic B, D, F vaccine immunization group is obviously higher than that of the duck DTMUV antibody blocking rate of the B, D, F vaccine control immunization group detected in the same period. The results are shown in tables 5 to 6 and FIGS. 6 to 7.
TABLE 5 inactivated vaccine and subunit vaccine respective test groups duck DTMUV ELISA antibody detection results (blocking rate PI%)
Note: "-" indicates that the antibody blocking rate was negative.
TABLE 6 live vaccine test results of duck DTMUV ELISA antibody (blocking rate PI%)
Note: "-" indicates that the antibody blocking rate was negative.
TABLE 7 inactivated vaccine and subunit vaccine challenge test results 14 days, 4 months, 5 months, 6 months after immunization
TABLE 8 live vaccine challenge test results at 14 days, 4 months, 5 months, and 6 months after immunization
The result of virus challenge in table 7 shows that the DTMU virus separation after virus challenge for 14 days after secondary immunization, the DTMU virus separation after virus challenge for the blank control group ducks without immunization is 10/10 positive, the DTMU virus separation after virus challenge for B, D vaccine immunization group ducks with IL-2 synergy is 10/10 negative, the protection rates are 100%, the DTMU virus separation after virus challenge for B, D immunization control group ducks is 1/10 positive and 2/10 positive respectively, and the protection rates are 90% and 80% respectively; 4 months after secondary immunization, the immune protection rates of the B, D vaccine immune group cooperated with the IL-2 are 100 percent, and the immune protection rates of the B, D immune control group are 80 percent and 70 percent respectively; 5 months after secondary immunization, the immune protection rate of an IL-2 synergistic B, D vaccine immune group is 100 percent, and the immune protection rate of a B, D immune control group is 10 percent; 6 months after the second immunization, the immunity protection rates of the B, D vaccine immunization groups cooperated with IL-2 are both 90%, and the immunity protection rates of the B, D immunization control groups are both 0;
as can be seen from the results of the challenge in table 8, the DTMU virus isolation after challenge for 21 days after immunization, and the DTMU virus isolation after challenge for the nonimmunized blank control group ducks were 10/10 positive, the DTMU virus isolation after challenge for the IL-2 synergistic F vaccine immunization group ducks was 10/10 negative, the protection rates were 100%, and the DTMU virus isolation rates after challenge for the F immunization control group ducks were 2/10 and 80%, respectively. 4 months after immunization, the immune protection rate of the F vaccine immune group with IL-2 synergy is 100%, and the immune protection rate of the F immune control group is 70%; 5 months after immunization, the immune protection rate of the F vaccine immune group with IL-2 synergy is 100%, and the immune protection rate of the F vaccine immune group is 10%; at 6 months after immunization, the immune protection rate of the F vaccine immune group cooperated with IL-2 is 80%, while the immune protection rate of the F immune control group is 0;
the virus attacking results of different time points show that the virus attacking protection rate of the IL-2 synergistic B, D, F vaccine immune group duck DTMU is far higher than that of B, D, F immune control group duck DTMU virus which is detected at the same time. And the immune protection period of the B, D, F vaccine of the synergetic immunization group is remarkably prolonged until the protection rate is not lower than 80% after 6 months of immunization, while the immune protection rate of the B, D and F vaccine of the immunization control group is remarkably reduced after 4 months of immunization until 5 months and 6 months of immunization have no protection force basically.
Example 6 test of Effect of immunopotentiator IL-2 on Immunity
In order to evaluate the immune function of the IL-2 to the organism, the cell level indexes of lymphocytes, macrophages, NK cells and the like are measured, and the result shows that the IL-2 can obviously improve the lymphocyte stimulation index of the organism, promote the phagocytic capacity of the macrophages and the killing capacity of the NK cells, and shows that the IL-2 has good immune enhancement effect.
(1) 20 test ducks are randomly divided into 2 groups of 10 ducks, one group is used as a test group to be injected with 0.5ml of IL-2 injection, and the other group is used as a control group to be injected with 0.5ml of normal saline. Collecting blood 21 days after injection, separating lymphocytes, and regulating cell suspension concentration to 1.0 × 107And/ml, added to a 96-well cell culture plate, 100 μ l/well, test group ducks and group ducks were set with test groups of + ConA group and-ConA group, respectively, two replicate wells per group, + ConA well: add ConA 10. mu.l and cell culture broth 90. mu.l, -ConA group: the cell culture medium was added in an amount of 100. mu.l, and the schematic drawing of the test addition is shown in FIG. 8. Culturing for 44 hours in an incubator, removing the cell culture plate, adding 10 mu l of MTT solution, mixing uniformly, continuing culturing for 4 hours, discarding the supernatant, adding DMSO to lyse the cells, shaking for 5min by a micro-shaker, and determining the OD570 value of the cells by an enzyme linked immunosorbent assay detector after the precipitate is completely dissolved. Calculated according to a calculation formula of lymphocyte stimulation indexesLymphocyte Stimulation Index (SI) per duck, wherein SI is test well OD 570/control well OD 570;
note: A1-A10: 10 test group ducks + ConA wells; B1-B10 are A1-A10 repeated holes;
C1-C10: 10 test group ducks-ConA wells; D1-D10 are C1-C10 repeat holes;
E1-E10: 10 control group ducks + ConA wells; F1-F10 are E1-E10 repeated holes;
G1-G10: 10 test group ducks-ConA wells; H1-H10 are G1-G10 repeat holes;
A11-D11: a cell control well;
A12-D12: MTT + lysate control.
Results show that the lymphocyte Stimulation Indexes (SI) of the duck in the test group injected with the IL-2 preparation are far higher than those of the duck in the control group, the IL-2 preparation can obviously improve the lymphocyte stimulation indexes of the body and can improve the cellular immune efficacy, and the specific results are 10.
TABLE 10 lymphocyte Stimulation Index (SI) value results
(2) Macrophage index detection
20 test ducks are randomly divided into 2 groups, each group comprises 10 ducks, one group is used as a test group to be injected with 0.5ml of IL-2 injection, and the other group is used as a control group to be injected with 0.5ml of normal saline. On 21 days after injection, the wings of the two groups of test ducks were injected into the veins of 1: 3 diluting India ink 0.2mL, timing immediately, collecting blood from heart 0.2mL 2min and 10min after ink injection, respectively, adding 20mL 0.1% Na2CO3Shaking the solution evenly, and using Shimadzu ultraviolet spectrophotometer
UVmini-1240 optical density values were measured at a wavelength of 600nm, as Na2CO3The solution was used as a blank control. Bleeding and killing the carotid artery of the chicken, weighing the liver and the spleen, and calculating the phagocytosis index.
Phagocytosis index ═ body weight ÷ (liver weight + spleen weight) × K1/3;
k is carbon clearance rate, and K is (lgOD1-lgOD 2)/T2-T1;
OD1 and OD2 are optical density values of blood to be measured obtained by blood sampling at 2min and 10min respectively; t1 and T2 indicate blood collection time after India ink injection.
Results the macrophage indexes (SI) of the ducks in the test group injected with the IL-2 preparation are far higher than those of the ducks in the control group, which shows that the IL-2 preparation can obviously improve the phagocytic capacity of macrophages of organisms and can improve the cellular immunity effect.
TABLE 10 macrophage index (SI) value results
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> Beijing Baoyi Biotechnology Co., Ltd, Beijing Yinong Biotech Co., Ltd, Hebei Baoying Biotechnology Co., Ltd
<120> duck interleukin-2 for immunopotentiation and use thereof as immunopotentiator
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Leu Lys Pro Val Thr Thr Asn Ala Gly Glu Leu Ile Gly Gln Leu Met
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Ala Tyr Asp Gly Leu Glu Lys Gln Ala Gly Glu Ser Lys Gly Ser Ser
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Ser Ser Ala Pro Leu Ser Glu Lys Asp Asn Thr Leu Thr Thr Leu Ile
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Lys Asp Leu Glu Asn Leu Gly Thr Ser Met Asn Gly Ile Asp Leu Glu
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Leu Tyr Thr Pro Asn Asp Thr Lys Glu Cys Ser Trp Gln Thr Leu Gln
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Asp Glu Ile Glu Asp Glu Asn Val Ser Ser Val Arg Asn Ile Lys Met
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Ile Cys Glu Ala Asn Ala Lys Asn Phe Pro Glu Phe Arg Arg Glu Leu
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Thr Asn Phe Leu Arg Ser Met Leu Lys
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accactctga taaaagacct agaaaacctg ggaacaagca tgaacgggat cgatcttgag 240
ctctacacac caaatgacac aaaggagtgc tcttggcaaa ctctgcaatg ttacttgaaa 300
gaaatagtca ccttggagaa agaaatcgaa gatgaagatg aaattgaaga tgagaacgta 360
tcttctgttc ggaatatcaa aatgaacctg cagaaactta tggacctaat tcccccaaga 420
accggttgca acatctgcga agctaatgcc aagaacttcc ccgaatttcg tcgtgaactg 480
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Claims (10)
1. A duck interleukin-2 protein for immune enhancement, the duck interleukin-2 protein being (a1) or (a2) as follows:
(a1) a protein consisting of amino acid residues shown in a sequence 1 in a sequence table;
(a2) and (a1) derived protein which is obtained by substituting and/or deleting and/or adding (a1) by one or more amino acid residues and has the function of the immune-enhanced duck interleukin.
2. A nucleic acid molecule encoding the protein of claim 1.
3. The nucleic acid molecule of claim 2, wherein the nucleic acid molecule is a DNA molecule according to any one of (b1) to (b3) below:
(b1) the nucleotide sequence is a DNA molecule of a sequence 2 in a sequence table;
(b2) a DNA molecule having 75% or more identity to the nucleotide sequence defined in (b1) and encoding the protein of claim 1;
(b3) a molecule that hybridizes under stringent conditions to the nucleotide sequence defined in (b1) and encodes the protein of claim 1.
4. An expression cassette, recombinant vector, recombinant microorganism or transgenic cell line comprising the nucleic acid molecule of claim 2 or 3.
5. The recombinant microorganism according to claim 4, wherein the nucleic acid molecule according to claim 2 or 3 is introduced into E.coli strain BL21(DE3) to obtain a recombinant Escherichia coli.
6. The recombinant Escherichia coli BYSW007 as claimed in claim 5, which is deposited in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the deposition number of CGMCC NO. 22853.
7. Use of recombinant Escherichia coli BYSW007 as claimed in claim 6 for the preparation of a protein as claimed in claim 1.
8. The use of the protein of claim 1, which is at least one of (c1) - (c8) as follows:
(c1) as an immunopotentiator;
(c2) preparing an immunopotentiator;
(c3) as an immunopotentiator for vaccines;
(c4) as an immunopotentiator for duck tembusu virus vaccines;
(c5) preparing a vaccine;
(c6) preparing a duck tembusu virus vaccine;
(c7) as an antiviral agent;
(c8) preparing the antiviral preparation.
9. The use according to claim 8,
the immunopotentiator comprises the following components: each milliliter of the feed contains 10000IU of duck interleukin-2 protein, 1-5 mg of achyranthes polysaccharide and 50-200 mug of vitamin C; 5 ng-10 ng of levamisole, and the balance of phosphate buffer solution.
10. A method for preparing the duck interleukin-2 protein for immune enhancement of claim 1, comprising the steps of: culturing the recombinant Escherichia coli BYSW007 of claim 6 to obtain the duck interleukin-2 protein.
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