Patent Application
20190276512
The present invention relates to a recombinant chicken interleukin-1β protein, and in particular, to a recombinant chicken interleukin-1β protein for producing antibody early and retaining for a longer period of time
The poultry industry in global continues to show impressive growth year by year. Therefore, the poultry farm in developed areas use a high density confinement rearing method to increase breeding density, which causes an urgent problem, government agency should spend more effort into biosecurity and disease prevention. In recent years, some countries have been faced the difficult challenges of a dramatic increase in the incidence of infectious disease outbreaks, some result from poor hygiene and management, but most of which result from a highly aggressive strain of virus, such as avian infectious bronchitis, infectious bursal disease, Newcastle disease and avian influenza, etc. In 2005, there were outbreaks of avian influenza in southern Taiwan, which not only caused a lot of deaths in ducks and geese, but also endangered human lives and resulted in huge economic impact and financial crisis. The best way to prevent infection with avian influenza is to avoid sources of exposure whenever possible. Most bird infections with avian influenza in poultry farms will be culled to avoid the viruses spreading. However, in high-density poultry farms, once the high-pathogenic avian influenza outbreaks, the speed of culling the infected birds may be less than that of the high-pathogenic avian influenza spread. Therefore, administrating a high-quality vaccine or vaccine adjuvant is the most effective way to prevent infection and severe outcomes caused by influenza viruses. And it needs to step up the preventive measures against avian influenza.
For animal vaccines, there are five poultry vaccines against Newcastle disease, avian infectious bronchitis, infectious bursal disease and fowl pox, which occupies 70 percent in vaccine testing, one third of the live attenuated fowl pox virus vaccines is produced in Taiwan, nearly 90 percent of the remaining vaccines are imported from foreign countries. Moreover, the adjuvant in a high titer avian vaccine is only obtained from foreign countries, which leads to higher prices and the lack of market competitiveness.
Recently, two main known vaccine adjuvants, aluminum-based and oil-based, are used in global livestock. The adverse effect of both vaccine adjuvants is chemical-makeup, wherein the aluminum-based adjuvant cannot enhance the specificity of the Th1 cell immune response. Oil-based adjuvant can trigger immune system response, but it may result in local inflammation and granulomatous reactions at the site of injection, chronic inflammation, skin ulceration, local abscess or tissue sloughing, diffuse systemic granulomas, and it is also unable to enhance the production of antibodies.
As such, the present invention provides a recombinant chicken interleukin-1β protein for producing antibody early and retaining for a longer period of time, which is created by point mutation in a genetic engineering method. The recombinant chicken interleukin-1β protein of the present invention can significantly improve the original vaccine efficacy to enhance antibody responses, to produce antibody one week earlier and to extend the protective effect until chickens sold off, and can produce significant higher antibody responses than the with-type chicken interleukin-1β protein.
A primary objective of the present invention is to provide a recombinant chicken interleukin-1β protein, which is encoded by the nucleotide sequence of SEQ ID NO:2.
Another objective of the present invention is to provide a method of producing antibody early and retaining for a longer period of time by using the recombinant chicken interleukin-1β protein.
A further objective of the present invention is to provide a method of producing antibody early and retaining for a longer period of time by using a vaccine supplemented with the recombinant chicken interleukin-1β protein.
According to an embodiment of the present invention, a time for producing antibody is one week earlier
According to an embodiment of the present invention, the longer period of time is at least four weeks to extend the protective effect, and a booster is not given.
According to an embodiment of the present invention, the recombinant chicken interleukin-1β protein is a component of a vaccine.
According to an embodiment of the present invention, the vaccine is an inactivated vaccine or an attenuated vaccine.
According to an embodiment of the present invention, the recombinant chicken interleukin-1β protein is a component of a vaccine adjuvant.
Accordingly, the recombinant chicken interleukin-1β protein of the present invention as a biological adjuvant directly use with an inactivated or activated avian vaccine used in the veterinary vaccines market, it can significantly improve the original vaccine efficacy to enhance antibody responses, and to produce neutralizing antibody against virus earlier. The recombinant chicken interleukin-1β protein of the present invention helps to develop avian interleukin-1β vaccine adjuvant and uses in medical application and livestock production.
The present invention will be apparent to those skilled in the art by reading the following detailed description of a preferred embodiment thereof, with reference to the attached drawings, in which:
The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification. The drawings illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.
The present invention provides a recombinant chicken interleukin-1β protein for producing antibody early and retaining for a longer period of time, which can stimulate cytokine secretion and enhance antibody production. The recombinant chicken interleukin-1β protein is an effective and low cost vaccine adjuvant for outbreaks of avian disease.
Therefore, the present invention constructs two new recombinant chicken interleukin-1β protein from an improved design of the amino acid sequence of wild-type chicken interleukin-1β (IL-1β), which are respectively named as the recombinant chicken interleukin-1β protein having first mutation (ChIL-1β mutant 1) and the recombinant chicken interleukin-1β protein having second mutation (ChIL-1β mutant 2). The present invention uses the recombinant chicken interleukin-1β proteins with a common vaccine in Taiwan and global poultry industry, Newcastle disease (ND), to conduct animal clinical trials. The results show that the recombinant chicken interleukin-1β protein of the present invention can stimulate the immune system to produce antibody for ND vaccine, and the chicken can only receive one dose to continue producing antibodies and to extend the protective effect. The local tissue administrated vaccine or vaccine adjuvant comprising the recombinant chicken interleukin-1β protein not only can produce secretory immunoglobulin A (IgA) but also maintain a high level of immunoglobulin G (IgG) in the blood. In addition, the vaccine comprising the recombinant chicken interleukin-1β protein can have positive synergistic effect on high IgG antibody production rates to extend the protective effect until chickens sold off. Therefore, the recombinant chicken interleukin-1β protein of the present invention helps to develop avian vaccine adjuvant and uses in medical application and livestock production.
The flow chart of constructing the circular permutation interleukin-1β (CP IL-1β) in one embodiment of the present invention as shown in
First, designing the forward and reverse primers comprising the point mutation for the two point mutation sequences of chicken interleukin-1β protein (SEQ ID NO:2 and SEQ ID NO: 3), wherein Q19A forward and reverse primers of the first mutation sequence (SEQ ID NO:2) respectively are SEQ ID NO: 4 and SEQ ID NO: 5; R140A forward and reverse primers of the second mutation sequence (SEQ ID NO:3) respectively are SEQ ID NO:6 and SEQ ID NO:7. And, using polymerase chain reaction to amplify wild-type chicken interleukin-1β sequence (SEQ ID NO:1) for the mutation site, wherein wild-type chicken interleukin-1β sequence is as a template (SEQ ID NO:1), the forward primer of wild-type chicken interleukin-1β (SEQ ID NO:8) and Q19A reverse primer (SEQ ID NO: 5) amplify Q19A mutation site to obtain the first fragment of the first mutation sequence (SEQ ID NO: 10), Q19A forward primer (SEQ ID NO:4) and the reverse primer of wild-type chicken interleukin-1β (SEQ ID NO: 9) amplify Q19A mutation site to obtain the second fragment of the first mutation sequence (SEQ ID NO: 11); and the forward primer of wild-type chicken interleukin-1β (SEQ ID NO:8) and R140A reverse primer (SEQ ID NO: 7) amplify R140A mutation site to obtain the first fragment of the second mutation sequence (SEQ ID NO: 12), R140A forward primer (SEQ ID NO:6) and the reverse primer of wild-type chicken interleukin-1β (SEQ ID NO: 9) amplify R140A mutation site to obtain the second fragment of the second mutation sequence (SEQ ID NO: 13). Then, the first (SEQ ID NO: 10) and second (SEQ ID NO: 11) fragments of the first mutation sequence serve as templates and primers for extension to obtain the first mutation of the recombinant chicken interleukin-1β sequence having Q19A mutation site (SEQ ID NO:2); and the first (SEQ ID NO: 12) and second (SEQ ID NO: 13) fragments of the second mutation sequence serve as templates and primers for extension to obtain the second mutation of the recombinant chicken interleukin-1β sequence having R140A mutation site (SEQ ID NO:3).
Furthermore, amplifying the first mutation of the recombinant chicken interleukin-1β sequence having Q19A (SEQ ID NO:2) as template using the forward (SEQ ID NO:8) and reverse (SEQ ID NO:9) primers of wild-type chicken interleukin-1β; and amplifying the second mutation of the recombinant chicken interleukin-1β sequence having R140A mutation site (SEQ ID NO:3) as template using the forward (SEQ ID NO:8) and reverse (SEQ ID NO:9) primers of wild-type chicken interleukin-1β.
Finally, cloning, expressing and purifying wild-type chicken interleukin-1β sequence (SEQ ID NO: 1), the first mutation of the recombinant chicken interleukin-1β sequence (SEQ ID NO: 2), and the second mutation of the recombinant chicken interleukin-1β sequence (SEQ ID NO: 3). As shown in
To determine the in vivo activity of the recombinant chicken interleukin-1β, the present invention detects the plasma cortisol level after the recombinant chicken interleukin-1β proteins having the first (ChIL-1β mutant 1) and the second (ChIL-1β mutant 2) mutation are respectively injected into the wing vein of specific pathogen free (SPF) chicken. As shown in
To determine vaccine adjuvant effect of the recombinant chicken interleukin-1β protein, the present invention administrates Newcastle disease (ND) live attenuated vaccine or which supplemented with the recombinant chicken interleukin-1β protein to specific pathogen free (SPF) chickens via eye drop, the chickens at one week of age is administrated primary (PRI) dose, the chickens at three weeks of age is administrated booster dose. The present invention draws blood samples from the wing vein of the chickens at two and four weeks of age to evaluate the immune response to Newcastle disease vaccine by measuring HI antibody titers using hemagglutination inhibition test.
As shown in
To evaluate the humoral immunity response produced by the host administrated the recombinant chicken interleukin-1β as vaccine adjuvant, the present invention performs a 1:100 dilution of the serum obtained from EXAMPLE 3 to measure the quantity of avian IgG using enzyme-linked immunosorbent assay (ELISA).
As shown in
To confirm the IgA distribution secreted by mucosal immune system in the nasal cavity of the host after administrating ND live attenuated vaccine supplemented with the recombinant chicken interleukin-1β protein via nose drop. The nasal cavity tissues are cut into slices three weeks after vaccination and stained using immunohistochemistry.
As show in
To test the protective effect of only administrating host one dose of ND live attenuated vaccine supplemented with the recombinant chicken interleukin-1β protein. The present invention follows EXAMPLE 3 protocol to administrate host at one week of age one dose of ND vaccine supplemented with the vaccine adjuvant of the recombinant chicken interleukin-1β protein having the second mutation (ChIL-1β mutant 2), and draws blood samples of host at two and four weeks of age to evaluate the immune response by measuring HI antibody titers using hemagglutination inhibition test.
As shown in
Not all Mutant Chicken IL-1β Proteins have Better Vaccine Adjuvant Effects than Those of Wild-Type Chicken IL-1β Proteins
In order to confirm that not all mutant chicken IL-1β proteins, as Q19A and R140A, have better vaccine adjuvant effects than those of wild-type chicken IL-1β proteins, novel mutant chicken IL-1β_T7A and IL-1β_E118R genes are designed. Gene cloning, recombinant protein expression, mutant chicken recombinant protein purification, and in vivo activity analysis were performed. Further, as the ND vaccine adjuvant and mixed vaccine immunized chicken, blood drawing per week for three weeks after immunization was performed to monitor the relative titer of antibodies against ND virus. The results show that the biological activity of mutant chicken IL-1β_T7A is similar to that of wild-type chicken IL-1β, and the effect as an NDV vaccine adjuvant of mutant chicken IL-1β_T7A is similar to that of wild-type chicken IL-1β. More interestingly, mutant chicken IL-1β_E118R not only has almost no biological activity, but it is almost ineffective as an NDV vaccine adjuvant. These results indicate that not all mutant chicken IL-1β proteins that have single point mutation on amino acid have better vaccine adjuvant effects than those of wild-type chicken IL-1β proteins.
First, the genetic recombination cloning method was constructed. The primers required for the amplification of the mutant chicken IL-1β DNA were designed, and submitted to MDBio, Inc. for sequence synthesis (Table 2). These designs are designed to mutate the seventh amino acid residue of threonine (T) into alanine (A) in the amino acid sequence of chicken and the newly constructed mutant gene is called ChIL-1β_T7A (SEQ ID NO: 14). In addition, the 118th amino acid residue of glutamate was replaced by arginine (R), and the newly constructed mutant gene name is called ChIL-1β_E118R (SEQ ID NO: 17).
First, wild-type chicken IL-1β (ChIL-1β) and mutant ChIL-1βT7A and E118R, which are newly synthesized and amplified, were genetically selected, and then the correctness of the sequences were detected by DNA sequencing. The E. coli expression system was used to express and purify mutant chicken IL-1β recombinant proteins, and soluble proteins with high-yield and high-purity were successfully obtained. The molecular weight of the mutant chicken IL-1β recombinant proteins is similar to that of the wild-type chicken IL-1β protein, which is about 23.6 kDa (
To examine the biological activity of the designed mutant IL-1β in chickens, a plasma cortisol concentration analysis was performed in vivo. Phosphate buffer solution (PBS), the wild-type chicken IL-1β (ChIL-1β) protein, the mutant chicken IL-1β T7A (ChIL-1β mutant T7A) protein, and the mutant chicken IL-1β E118R (ChIL-1β mutant E118R) protein were injected intravenously into the wing vein of specific pathogen free (SPF) chicken, because functional chicken IL-1β induces the chicken central system to produce cortisol to enter the bloodstream to resist IL-1β. After three hours, the blood of the chickens under test was taken, and the relative plasma cortisol levels induced by the wild-type chicken IL-1β protein and mutant chicken IL-1β proteins were measured (
The result shows that the relative plasma cortisol level induced by ChIL-1β mutant T7A was similar to that of the wild-type chicken IL-1β, and the relative plasma cortisol level induced by ChIL-1β mutant E118R was significantly decreased, almost the same as the cortisol level in the central system induced by PBS in the negative control group. Compared with the relative plasma cortisol level induced by the wild-type chicken IL-1β, the relative plasma cortisol level induced by ChIL-1β mutant T7A and ChIL-1β mutant E118R was 82% and 10%, respectively.
That is, ChIL-1β mutant T7A has almost similar functionality to the wild-type chicken IL-1β, and the functionality is not improved due to point mutation on amino acid residues (threonine to alanine). On the contrary, the functionality is maintained as that of the wild-type chicken IL-1β. Further, ChIL-1β mutant E118R almost loses the functionality of chicken IL-1β, and the ability to induce cortisol production by ChIL-1β mutant E118R is lost as that of PBS in the negative control group. Therefore, not changing any amino acid residue on the wild-type chicken IL-1β can enhance the function of the wild-type chicken IL-1β. ChIL-1β mutant E118R is similar to PBS, but has little ability to induce cortisol (
In order to further understand whether chicken IL-1β or mutant chicken IL-1β proteins in which any amino acid residue alteration was constructed (e.g., ChIL-1β mutant T7A and ChIL-1β mutant E118R) can be used as a chicken vaccine adjuvant, chicken Newcastle disease (ND)-live vaccine (VOLVAC ND LaSota MLU, Boehringer Ingelheim Vetmedica, S.A. de C.V., Mexico) alone without chicken IL-1β (group 2), mixed with 1 μg of wild-type chicken IL-1β (group 3), ChIL-1β mutant T7A (group 4), or ChIL-1β mutant E118R (group 5) was vaccinated to one-week-old SPF chicks via nasal drop vaccination once. Another group of chicks as a negative control group were vaccinated with PBS via nasal drop vaccination. 0, 1, 2, and 3 weeks after vaccination, blood was drawn from the chicken wing vein, and these sera were subjected to anti-NDV hemagglutination inhibition (HI) test to detect the HI antibody titers against NDV when the wild-type chicken IL-1β and mutant chicken IL-1β were used as an NDV vaccine adjuvant (
The result indicates that the HI antibody titers of protective antibody produced by ChIL-1β mutant T7A (group 4) assisted NDV vaccine 1, 2, and 3 weeks after vaccination were similar to those of the wild-type chicken IL-1β (group 3), and the HI antibody titers of protective antibody produced by ChIL-1β mutant E118R (group 5) assisted NDV vaccine 1, 2, and 3 weeks after vaccination were similar to those of the NDV vaccine alone (
The present invention is to create two kinds of the recombinant chicken interleukin-1β protein as vaccine adjuvant using point mutation in a genetic engineering method, it can significantly enhance the capability of producing antibody, produce antibody one week earlier, and extend the protective effect until chicken sold off. Furthermore, the present invention has validated that the recombinant chicken interleukin-1β protein as a vaccine adjuvant using with Newcastle disease (ND) has significant effects on immune response. Also, the cost of the recombinant chicken interleukin-1β protein as a vaccine adjuvant is 0.1 Taiwan Dollar (TWD) for each chicken, it can be more lower cost resulted from mass production to produce market competition. The recombinant chicken interleukin-1β protein has a biological decomposition, and it can store at a room temperature or in the freezer to maintain activity for 4 to 10 days (not precipitation), and store at −20° C. to maintain activity for 6 months (not precipitation). Therefore, the recombinant chicken interleukin-1β protein has good quality, stability, safety and including no side effects.
Accordingly, the recombinant chicken interleukin-1β protein of the present invention as a biological adjuvant directly uses with an inactivated or activated avian vaccine used in the veterinary vaccines market, it can significantly improve the original vaccine efficacy to enhance antibody responses, produce neutralizing antibody against virus earlier. The recombinant chicken interleukin-1β protein of the present invention helps to develop avian interleukin-1β vaccine adjuvant and uses in medical application and livestock production. Nowadays, all countries in the world including Taiwan have faced the problems of drug-resistance updates and new mutant recombinant virus including avian influenza; therefore, the recombinant chicken interleukin-1β protein of the present invention as a vaccine adjuvant can significantly enhance the effect of vaccine to protect livestock and poultry from disease threats.
Although the present invention has been described with reference to the preferred embodiments thereof, it is apparent to those skilled in the art that a variety of modifications and changes may be made without departing from the scope of the present invention which is intended to be defined by the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 105109016 | Mar 2016 | TW | national |
This application is a continuation-in-part of U.S. patent application Ser. No. 15/153,704, filed on May 12, 2016, which claims the priority benefit of Taiwan patent application No. 105109016 filed on Mar. 23, 2016. The entirety of each of the above-mentioned patent applications is hereby incorporated by reference herein and made a part of this specification.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15153704 | May 2016 | US |
| Child | 16424693 | US |
