KR20230153038A - Recombinant vector comprising genes encoding RANKL protein and immunological adjuvant and feed additive for chicken using the same - Google Patents
Recombinant vector comprising genes encoding RANKL protein and immunological adjuvant and feed additive for chicken using the same Download PDFInfo
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- KR20230153038A KR20230153038A KR1020220052747A KR20220052747A KR20230153038A KR 20230153038 A KR20230153038 A KR 20230153038A KR 1020220052747 A KR1020220052747 A KR 1020220052747A KR 20220052747 A KR20220052747 A KR 20220052747A KR 20230153038 A KR20230153038 A KR 20230153038A
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- South Korea
- Prior art keywords
- lactic acid
- acid bacteria
- crankl
- recombinant
- cell extract
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Abstract
본 발명은 RANKL 단백질을 코딩하는 유전자를 포함하는 재조합 벡터 및 이것을 포함하는 면역증강용 보조제에 대한 것으로, 특히 치킨으로부터 유래한 cRANKL(chicken Receptor activator of nuclear factor-kB ligand) 단백질을 코딩하는 유전자를 포함하는 것을 특징으로 하여, 장내 M 세포의 분화를 활성화시키고 소장 내 항원을 흡수하는 점막면역반응을 유도하는데 탁월한 효과를 나타내었다. 이에 따라, 본 발명의 cRANKL을 포함하는 재조합 유산균을 경구 백신 보조제로 이용하면, 소장 점막면역반응을 효율적으로 유도하고 경구 백신의 소장점막에서 낮은 흡수 효율의 문제점을 해결함으로써, 경구 백신의 효과를 증진시킬 수 있다. 또한, 본 발명은 cRANKL이 포함된 재조합 유산균의 세포 추출물을, 특별히 양계용 경구 백신 보조제 또는 양계용 사료 첨가제로 이용할 수 있는 효과가 있다. The present invention relates to a recombinant vector containing a gene encoding the RANKL protein and an adjuvant for immune enhancement containing the same, particularly containing a gene encoding the cRANKL (chicken Receptor activator of nuclear factor-kB ligand) protein derived from chicken. Characterized by this, it showed an excellent effect in activating the differentiation of intestinal M cells and inducing a mucosal immune response that absorbs antigens in the small intestine. Accordingly, when the recombinant lactic acid bacteria containing cRANKL of the present invention are used as an oral vaccine adjuvant, the effectiveness of the oral vaccine is improved by efficiently inducing an immune response in the small intestine mucosa and solving the problem of low absorption efficiency in the small intestine mucosa of the oral vaccine. You can do it. In addition, the present invention has the effect of using the cell extract of recombinant lactic acid bacteria containing cRANKL, especially as an oral vaccine adjuvant for poultry or as a feed additive for poultry.
Description
본 발명은 RANKL 유전자를 포함하는 재조합 벡터에 대한 것으로, 특히 Chicken RANKL (cRANKL) 유전자를 포함하는 재조합 벡터, 상기 벡터로 형질 전환되어서 수용성 cRANKL을 생산하는 재조합 유산균, 상기 재조합 유산균을 배양한 배양물로부터 얻은 세포 추출물, 및 이것을 포함하는 면역증강용 보조제와 경구 백신 보조제 및 사료 첨가제에 대한 것이다. The present invention relates to a recombinant vector containing the RANKL gene, in particular, a recombinant vector containing the Chicken RANKL (cRANKL) gene, a recombinant lactic acid bacteria that is transformed with the vector to produce water-soluble cRANKL, and a culture in which the recombinant lactic acid bacteria are cultured. It relates to the obtained cell extract, and adjuvants for enhancing immunity, oral vaccine adjuvants, and feed additives containing the same.
경구백신은 주사로 인한 스트레스가 적을 뿐만 아니라, 경구백신을 통해 점막면역반응이 활성화되면, 분비형 항체인 IgA가 소장 내강에서 병원균을 무력화시켜 병원균이 소장 상피세포를 통과하여 체내로 들어오지 못하도록 막아주기 때문에 병원균을 초도에 방어할 수 있다는 장점이 있다. 따라서 이질, 콜레라 등의 세균 및 바이러스성 수인성 전염병을 감염 경로상에서 직접적으로 차단하고 예방하기 위해서는 주사방식의 백신보다 경구백신을 통해 소화장관 내 점막면역반응을 활성화시키는 것이 효과적이다.Oral vaccines not only reduce the stress caused by injection, but when the mucosal immune response is activated through the oral vaccine, IgA, a secretory antibody, neutralizes pathogens in the small intestine lumen, preventing pathogens from entering the body through small intestine epithelial cells. Therefore, it has the advantage of being able to defend against pathogens at the first stage. Therefore, in order to directly block and prevent bacterial and viral waterborne infectious diseases such as dysentery and cholera along the infection route, it is more effective to activate the mucosal immune response in the digestive tract through an oral vaccine rather than an injectable vaccine.
그러나 백신체의 주요 구성요소인 단백질 등은 운송과정 중 위산에 의한 낮은 pH, 소장장관의 각종 영양소 분해효소 등에 의해 변성되어 그 고유 기능을 잃기 쉬우며, 안전하게 소화장관까지 전달되더라도 소장점막층에서의 흡수효율이 낮아 체내로 흡수되는데 문제점을 나타냄에 따라, 현재까지는 주사형 백신보다 효율성이 낮아 경구 백신의 활용이 매우 제한적이다.However, proteins, which are the main components of the vaccine, are easily denatured by the low pH caused by stomach acid and various nutrient-decomposing enzymes in the small intestine during the transportation process and lose their original functions. Even if they are safely delivered to the digestive tract, they are absorbed in the small intestine mucosa layer. As the efficiency is low and there are problems with absorption into the body, the use of oral vaccines is very limited to date because they are less efficient than injectable vaccines.
또한 전 세계적으로 판매되고 있는 경구백신은 10가지 미만으로 대부분 약독화된 생백신이다. 이러한 약독화 생백신은 병원체의 독성을 약화시키더라고 면역력이 약한 개체내에서는 백신이 다시 독성을 나타냄으로 잠재적인 위험요소가 될 수 있으며, 소장 점막에서는 점막면역반응의 반대 기작인 경구 관용(Oral tolerance)반응을 유도하여 오히려 병원균에 대한 면역시스템의 관용으로 생명체가 위험에 빠질 수 있다. 특히, 항원 단백질로 구성된 서브유닛(Subunit) 백신은 경구 관용 부작용의 문제가 알려짐에 따라, 점막 면역반응을 강하게 자극시킬 수 있는 경구 백신 면역증강제(Oral vaccine adjuvant)의 개발이 요구되고 있다.Additionally, there are less than 10 oral vaccines sold worldwide, and most of them are live attenuated vaccines. Even though these live attenuated vaccines attenuate the virulence of the pathogen, they can be a potential risk factor as the vaccine can become toxic again in individuals with weak immune systems, and oral tolerance, which is the opposite mechanism of the mucosal immune response, in the small intestine mucosa By inducing a response, the organism may be put at risk due to tolerance of the immune system to pathogens. In particular, as the problem of oral tolerance side effects has become known for subunit vaccines composed of antigen proteins, there is a need for the development of oral vaccine adjuvants that can strongly stimulate mucosal immune responses.
특히, 양계 산업에서 감염성 질병은 큰 손실을 가져온다. 사육환경이 열악하여 발생되는 질병들이 많은 것으로 알려져 있고, 특히 고병원성 조류 인플루엔자(AI) 바이러스가 전국적으로 대유행하면서 많은 가금류들이 살처분 되었다. In particular, infectious diseases cause great losses in the poultry industry. It is known that many diseases are caused by poor breeding environments, and in particular, as the highly pathogenic avian influenza (AI) virus spread nationwide, many poultry were culled.
이와 관련하여, 대한민국 공개특허 제10-2016-0000949호(발명의 명칭 :RANKL를 생산하는 재조합 유산균 및 이의 용도)는 서열번호 4의 아미노산으로 표시되는 RANKL 단백질을 코딩하는 유전자를 포함하는 재조합 벡터를 개시하고 있다. 그러나, 여기서 사용한 상기 RANKL 유전자는 마우스로부터 얻은 것이어서, 그로부터 얻은 RANKL 단백질을 양계나 가금용 면역증강제로 사용하기에는 부적합하다는 단점이 있다. 또한, 상기 공개특허의 in vivo 면역실험에서는 재조합 유산균주 자체를 마우스에 경구 투여 했는데, 이 처럼 유전자변형생물체(Living Modified Organism, 이하 LMO)를 동물에게 직접 투여하는 경우 자연생태계를 파괴할 수 있고, 독성이 있는 숙주에서 생산되는 재조합 단백질은 반드시 정제 과정을 거쳐야 하는 단점이 있다. In this regard, Republic of Korea Patent Publication No. 10-2016-0000949 (title of the invention: Recombinant lactic acid bacteria producing RANKL and uses thereof) discloses a recombinant vector containing a gene encoding the RANKL protein represented by the amino acid of SEQ ID NO: 4. It is starting. However, since the RANKL gene used here was obtained from mice, the RANKL protein obtained therefrom has the disadvantage of being unsuitable for use as an immune enhancer for poultry or poultry. In addition, in the in vivo immune experiment of the above-mentioned published patent, the recombinant lactic acid bacteria strain itself was orally administered to mice. In this way, if living modified organisms (LMO) are administered directly to animals, the natural ecosystem can be destroyed, Recombinant proteins produced in toxic hosts have the disadvantage of having to undergo a purification process.
본 발명은 상기한 문제점을 해결하기 위한 것으로, 소장 점막의 M 세포를 활성화시키는 인자인 RANKL을 생산하는 재조합 유산균을 제작하고, RANKL이 포함된 유산균 및 이것의 세포 추출물(Cell extracts)을 경구 백신 보조제 및 사료 첨가제로 활용하는 것이 목적이다.The present invention is intended to solve the above problems, by producing recombinant lactic acid bacteria that produce RANKL, a factor that activates M cells in the small intestine mucosa, and by producing lactic acid bacteria containing RANKL and their cell extracts as oral vaccine adjuvants. The purpose is to use it as a feed additive.
상기한 목적을 달성하기 위한 본 발명의 일 실시형태는 서열번호 1의 아미노산으로 표시되고, Usp45 분비 시그널(Usp45 secretion signal)과 연결된 cRANKL(chicken Receptor activator of nuclear factor-kB ligand) 단백질을 코딩하는 유전자를 포함하는 재조합 벡터이다.One embodiment of the present invention for achieving the above object is a gene encoding a cRANKL (chicken receptor activator of nuclear factor-kB ligand) protein expressed by the amino acid of SEQ ID NO: 1 and linked to the Usp45 secretion signal. It is a recombinant vector containing.
여기서, 상기 재조합 벡터는 도 1의 개열지도를 갖는 것이 가능하다. Here, it is possible for the recombinant vector to have the cleavage map of Figure 1.
그리고, 상기 유전자는 C 말단에 6 히스티딘(Histidine) 태그를 더 가지는 것일 수 있다. Additionally, the gene may have 6 more histidine tags at the C terminus.
본 발명의 일 구현예는, 상기한 재조합 벡터로 형질도입된 재조합 유산균이다.One embodiment of the present invention is a recombinant lactic acid bacteria transduced with the above-described recombinant vector.
상기 재조합 유산균은 소장 M 세포 분화 인자인 RANKL (Receptor activator of nuclear factor kappa-B ligand)을 생산하는 것이 가능하다.The recombinant lactic acid bacteria are capable of producing RANKL (Receptor activator of nuclear factor kappa-B ligand), a small intestine M cell differentiation factor.
본 발명의 일 구현예는, 상기한 재조합 유산균을 배양한 배양물로부터 얻은 세포 추출물이다.One embodiment of the present invention is a cell extract obtained from a culture of the above-mentioned recombinant lactic acid bacteria.
상기 세포 추출물은 상기 배양물을 원심분리하여 배양상층액과 균체로 분리하고, 상기 분리한 균체를 파쇄한 후 원심분리해서 얻은 세포질 단백질이 용해된 상층액인 것일 수 있다. The cell extract may be a supernatant in which cytoplasmic proteins are dissolved, obtained by centrifuging the culture, separating the culture supernatant and bacterial cells, disrupting the separated bacterial cells, and then centrifuging.
본 발명의 일 구현예는, 상기한 재조합 유산균을 유효성분으로 함유하는 면역증강용 보조제이다.One embodiment of the present invention is an adjuvant for immune enhancement containing the above-described recombinant lactic acid bacteria as an active ingredient.
본 발명의 일 구현예는, 상기한 세포 추출물을 유효성분으로 함유하는 경구 백신 보조제이다.One embodiment of the present invention is an oral vaccine adjuvant containing the above-described cell extract as an active ingredient.
상기 경구 백신 보조제는 양계용인 것이 가능하다.It is possible that the oral vaccine adjuvant is for poultry use.
본 발명의 일 구현예는, 상기한 세포 추출물을 유효성분으로 함유하는 양계용 사료 첨가제이다.One embodiment of the present invention is a feed additive for poultry containing the above-described cell extract as an active ingredient.
상기 면역증강용 보조제는 소장 M 세포를 활성화시켜 경구백신의 흡수 효율을 증가시키는 것일 수 있다. The adjuvant for immune enhancement may increase the absorption efficiency of oral vaccines by activating small intestine M cells.
본 발명의 일 구현예는 상기한 재조합 유산균 및 경구백신을 유효성분으로 포함하는 경구백신용 조성물이다.One embodiment of the present invention is an oral vaccine composition containing the above-described recombinant lactic acid bacteria and an oral vaccine as active ingredients.
기타 실시예들의 구체적인 사항들은 상세한 설명 및 도면들에 포함되어 있다. Specific details of other embodiments are included in the detailed description and drawings.
이러한 본 발명은 치킨으로부터 유래한 cRANKL(chicken Receptor activator of nuclear factor-kB ligand) 단백질을 코딩하는 유전자를 포함하는 것을 특징으로 하여, 장내 M 세포의 분화를 활성화시키고 소장 내 항원을 흡수하는 점막면역반응을 유도하는데 탁월한 효과를 나타내었다. The present invention is characterized by including a gene encoding a cRANKL (chicken Receptor activator of nuclear factor-kB ligand) protein derived from chicken, thereby activating the differentiation of intestinal M cells and promoting a mucosal immune response that absorbs antigens in the small intestine. It showed an excellent effect in inducing.
이에 따라, 본 발명의 cRANKL을 포함하는 재조합 유산균을 경구 백신 보조제로 이용하면, 소장 점막면역반응을 효율적으로 유도하고 경구 백신의 소장점막에서 낮은 흡수 효율의 문제점을 해결함으로써, 경구 백신의 효과를 증진시킬 수 있다.Accordingly, when the recombinant lactic acid bacteria containing cRANKL of the present invention are used as an oral vaccine adjuvant, the effectiveness of the oral vaccine is improved by efficiently inducing an immune response in the small intestine mucosa and solving the problem of low absorption efficiency in the small intestine mucosa of the oral vaccine. You can do it.
또한, 본 발명은 cRANKL이 포함된 재조합 유산균의 세포 추출물을, 특별히 양계용 경구 백신 보조제로 이용할 수 있는 효과가 있다. In addition, the present invention has the effect of using the cell extract of recombinant lactic acid bacteria containing cRANKL as an oral vaccine adjuvant, especially for poultry.
또한, 본 발명에 의하면, 소장의 M세포 분화 능력 향상으로 인해 외부 항원에 대한 면역반응을 증가시켜서, 면역증강용 사료 첨가제로 활용이 가능하다.In addition, according to the present invention, the immune response to external antigens is increased due to the improvement of the M cell differentiation ability of the small intestine, and it can be used as a feed additive for immunity enhancement.
도 1은 본 발명의 일 실시예에 따라 USP45 분비 시그널과 Chicken 유래 RANKL 유전자를 포함하여 제작된 재조합 벡터 지도(A) 및 USP45 분비 시그널을 포함하지 않고 제작된 재조합 벡터 지도(B)이다.
도 2는 본 발명의 일 실시예에 따른 재조합 벡터로 형질 전환된 재조합 유산균 균주에서 발현된 cRANKL 단백질을 확인한 Western blot 결과이다(여기서, Lane 1: cRANKL이 포함된 재조합 유산균(L. lactis (pILPtuf.cRANKL))의 세포 추출물, Lane 2: 야생형 유산균(L. lactis IL1403)의 세포 추출물. Lane 3-5: 상용 His-tagged Calmodulin (18 kDa) 1.5ug, 1ug, 0.5ug.).
도 3은 야생형(Wild type) 유산균과 본 발명의 일 실시예에 따른 재조합 유산균의 성장율을 확인한 결과이다.
도 4는 본 발명의 일 실시예에 따른 재조합 유산균의 RANKL 생산량을 측정하기 위한 상용 Calmodulin의 standard curve이다.
도 5는 본 발명의 일 실시예에 따른 재조합 유산균에서 생성된 cRANKL의 생물학적 활성을 확인하기 위해, in vitro에서 RAW 264.7 세포에 cRANKL이 포함된 재조합 유산균 세포 추출물을 처리한 후 RAW 264.7 세포가 Osteoclast-like 세포로 분화하는지를 분석(Cell differentiation assay)한 과정을 나타낸 모식도이다.
도 6은 본 발명의 일 실시예에 따른 재조합 유산균에서 생성된 cRANKL의 생물학적 활성을 확인하기 위해, 상기 도 5의 모식도에서와 같이 cRANKL이 포함된 재조합 유산균 세포 추출물을 처리한 RAW 264.7 세포에서 추출한 RNA로 Real-time (RT) PCR을 진행하여 Osteoclast-like 세포 분화와 관련된 유전자인 Tartrate-resistant acid phosphatase (TRAP)의 발현 정도를 분석한 결과이다.
도 7은 본 발명의 일 실시예에 따라 4주령 female BLAB/c mouse model에서 cRANKL이 포함된 재조합 유산균 세포 추출물이 점막면역증진 효과를 확인하기 위해 수행된 in vivo 분석 모식도이다.
도 8은 도 7의 모식도에서와 같이 야생형 유산균 세포 추출물과 본 발명의 일 실시예에 따라 cRANKL이 포함된 재조합 유산균 세포 추출물이 경구 투여된 BALB/c mouse의 소장 내 M 세포의 GP2 (Glycoprotein 2) mRNA 발현량을 RT-PCR로 측정한 결과이다. GP2 (Glycoprotein 2)는 M 세포의 마커이다.
도 9는 본 발명의 일 실시예에 따라 1일령 ROSS 308 양계에서 cRANKL이 포함된 재조합 유산균 세포 추출물의 점막면역증진 효과를 확인하기 위해 수행된 in vivo 분석 모식도이다.
도 10A는 도 9의 모식도에서와 같이 본 발명의 일 실시예에 따른 cRANKL이 포함된 재조합 유산균 세포 추출물의 경구 투여 최적 Dose를 결정하기 위해, 여러가지 양의 cRANKL이 포함된 재조합 유산균의 세포 추출물이 경구 투여된 Hy-Line Chicken의 소장 내 M 세포의 ANXA5 (Annexin 5) mRNA 발현량을 RT-PCR로 측정한 결과이다. ANXA 5 (Annexin 5)는 M 세포의 마커이다.
도 10B는 도 9의 모식도에서와 같이 야생형 유산균 세포 추출물과 본 발명의 일 실시예에 따라 cRANKL이 포함된 재조합 유산균 세포 추출물이 경구 투여된 ROSS 308 Chicken의 소장 내 M 세포의 ANXA5 mRNA 발현량을 RT-PCR로 측정한 결과이다. ANXA 5 (Annexin 5)는 M 세포의 마커이다.
도 11은 도 9의 모식도에서와 같이 야생형 유산균 세포 추출물과 본 발명의 일 실시예에 따라 cRANKL이 포함된 재조합 유산균 세포 추출물이 경구 투여된 ROSS 308 Chicken에게 Infectious bursal disease (IBD) 백신을 경구 투여 후 항원 특이적 항체의 생산량을 비교한 결과이다.Figure 1 shows a recombinant vector map (A) produced including the USP45 secretion signal and the Chicken-derived RANKL gene and a recombinant vector map (B) produced without the USP45 secretion signal according to an embodiment of the present invention.
Figure 2 is a Western blot result confirming the cRANKL protein expressed in a recombinant lactic acid bacteria strain transformed with a recombinant vector according to an embodiment of the present invention (here, Lane 1: recombinant lactic acid bacteria ( L. lactis (pILPtuf. cRANKL), Lane 2: Cell extract of wild-type lactic acid bacteria ( L. lactis IL1403). Lanes 3-5: Commercial His-tagged Calmodulin (18 kDa) 1.5ug, 1ug, 0.5ug.).
Figure 3 shows the results of confirming the growth rate of wild type lactic acid bacteria and recombinant lactic acid bacteria according to an embodiment of the present invention.
Figure 4 is a standard curve of commercial Calmodulin for measuring RANKL production of recombinant lactic acid bacteria according to an embodiment of the present invention.
Figure 5 shows that in order to confirm the biological activity of cRANKL produced from recombinant lactic acid bacteria according to an embodiment of the present invention, RAW 264.7 cells were treated with a recombinant lactic acid bacteria cell extract containing cRANKL in vitro, and then RAW 264.7 cells showed Osteoclast- This is a schematic diagram showing the process of analyzing whether cells differentiate into like cells (Cell differentiation assay).
Figure 6 shows RNA extracted from RAW 264.7 cells treated with recombinant lactic acid bacteria cell extract containing cRANKL as shown in the schematic diagram of Figure 5 to confirm the biological activity of cRANKL produced from recombinant lactic acid bacteria according to an embodiment of the present invention. This is the result of analyzing the expression level of tartrate-resistant acid phosphatase (TRAP), a gene related to osteoclast-like cell differentiation, by performing real-time (RT) PCR.
Figure 7 is a schematic diagram of an in vivo analysis performed to confirm the effect of recombinant lactic acid bacteria cell extract containing cRANKL in enhancing mucosal immunity in a 4-week-old female BLAB/c mouse model according to an embodiment of the present invention.
Figure 8 shows GP2 (Glycoprotein 2) of M cells in the small intestine of a BALB/c mouse to which a wild-type lactic acid bacteria cell extract and a recombinant lactic acid bacteria cell extract containing cRANKL according to an embodiment of the present invention were orally administered, as shown in the schematic diagram of Figure 7. This is the result of measuring the mRNA expression level by RT-PCR. Glycoprotein 2 (GP2) is a marker for M cells.
Figure 9 is a schematic diagram of an in vivo analysis performed to confirm the mucosal immunity enhancing effect of a recombinant lactic acid bacteria cell extract containing cRANKL in 1-day-old ROSS 308 chickens according to an embodiment of the present invention.
10A is a schematic diagram of FIG. 9, in order to determine the optimal dose for oral administration of a recombinant lactic acid bacteria cell extract containing cRANKL according to an embodiment of the present invention, cell extracts of recombinant lactic acid bacteria containing various amounts of cRANKL are administered orally. This is the result of measuring ANXA5 (Annexin 5) mRNA expression level in M cells in the small intestine of administered Hy-Line Chicken by RT-PCR. ANXA 5 (Annexin 5) is a marker of M cells.
Figure 10B shows the expression level of ANXA5 mRNA in M cells in the small intestine of ROSS 308 Chicken orally administered with a wild-type lactic acid bacteria cell extract and a recombinant lactic acid bacteria cell extract containing cRANKL according to an embodiment of the present invention as shown in the schematic diagram of Figure 9 by RT. -This is a result measured by PCR. ANXA 5 (Annexin 5) is a marker of M cells.
Figure 11 shows the wild-type lactic acid bacteria cell extract as shown in the schematic diagram of Figure 9 and the recombinant lactic acid bacteria cell extract containing cRANKL according to an embodiment of the present invention after oral administration of the Infectious bursal disease (IBD) vaccine to ROSS 308 Chicken. This is the result of comparing the production amount of antigen-specific antibodies.
본 발명은 다양한 변환을 가할 수 있고 여러 가지 실시 예를 가질 수 있는 바, 특정 실시 예들을 도면에 예시하고 상세한 설명에서 상세하게 설명하고자 한다. 그러나, 이는 본 발명을 특정한 실시 형태에 대해 한정하려는 것이 아니며, 본 발명의 사상 및 기술 범위에 포함되는 모든 변환, 균등물 내지 대체물을 포함하는 것으로 이해되어야 한다. 본 발명을 설명함에 있어서 관련된 공지 기술에 대한 구체적인 설명이 본 발명의 요지를 흐릴 수 있다고 판단되는 경우 그 상세한 설명을 생략한다.Since the present invention can be modified in various ways and can have various embodiments, specific embodiments will be illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and should be understood to include all transformations, equivalents, and substitutes included in the spirit and technical scope of the present invention. In describing the present invention, if it is determined that a detailed description of related known technologies may obscure the gist of the present invention, the detailed description will be omitted.
본 출원에서 사용한 용어는 단지 특정한 실시예를 설명하기 위해 사용된 것으로, 본 발명을 한정하려는 의도가 아니다. 단수의 표현은 문맥상 명백하게 다르게 뜻하지 않는 한, 복수의 표현을 포함한다. 본 출원에서, "포함하다" 또는 "가지다" 등의 용어는 명세서상에 기재된 특징, 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것이 존재함을 지정하려는 것이지, 하나 또는 그 이상의 다른 특징들이나 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것들의 존재 또는 부가 가능성을 미리 배제하지 않는 것으로 이해되어야 한다.The terms used in this application are only used to describe specific embodiments and are not intended to limit the invention. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this application, terms such as “comprise” or “have” are intended to designate the presence of features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, but are not intended to indicate the presence of one or more other features. It should be understood that this does not exclude in advance the possibility of the existence or addition of elements, numbers, steps, operations, components, parts, or combinations thereof.
제1, 제2 등의 용어는 다양한 구성요소들을 설명하는데 사용될 수 있지만, 상기 구성요소들은 상기 용어들에 의해 한정되어서는 안 된다. 상기 용어들은 하나의 구성요소를 다른 구성요소로부터 구별하는 목적으로만 사용된다. Terms such as first, second, etc. may be used to describe various components, but the components should not be limited by the terms. The above terms are used only for the purpose of distinguishing one component from another.
본 발명은 RANKL 유전자를 포함하는 재조합 벡터에 대한 것으로, 특히 Chicken RANKL (cRANKL) 유전자를 포함하는 재조합 벡터이다.The present invention relates to a recombinant vector containing the RANKL gene, particularly a recombinant vector containing the Chicken RANKL (cRANKL) gene.
본 발명의 일 실시형태는 서열번호 1의 아미노산으로 표시되고, Usp45 분비 시그널(Usp45 secretion signal)과 연결된 cRANKL(chicken Receptor activator of nuclear factor-kB ligand) 단백질을 코딩하는 유전자를 포함하는 재조합 벡터이다.One embodiment of the present invention is a recombinant vector containing a gene encoding a cRANKL (chicken receptor activator of nuclear factor-kB ligand) protein expressed by the amino acid of SEQ ID NO: 1 and linked to the Usp45 secretion signal.
즉, 본 발명에 따른 재조합 벡터는 서열번호 1의 아미노산으로 표시되는 단백질 또는 유전자를 포함하는 것이고, 상기 단백질은 Usp45 분비 시그널과 연결된 cRANKL 인 것이다. That is, the recombinant vector according to the present invention contains a protein or gene represented by the amino acid of SEQ ID NO: 1, and the protein is cRANKL linked to the Usp45 secretion signal.
치킨으로부터 유래된 cRANKL의 발현을 위하여, 신호 단백질로 Usp45 분비 시그널이 상기 cRANKL의 N 말단에 연결된 것이고, 상기 cRANKL는 치킨으로부터 유래되어 서열번호 1의 아미노산에 포함된 서열을 갖는 것이 특징이다. For the expression of cRANKL derived from chicken, the Usp45 secretion signal as a signal protein is linked to the N terminus of cRANKL, and cRANKL is derived from chicken and is characterized by having a sequence included in the amino acid of SEQ ID NO: 1.
후술하는 실시예에서 확인할 수 있는 바와 같이, USP45 분비 시그널이 없는 경우에는 벡터가 만들어지지 않았고, 유산균에 transformation 시켰을 때 mutation이 발생하였다.As can be seen in the examples described later, in the absence of the USP45 secretion signal, the vector was not created, and mutations occurred when transformed into lactic acid bacteria.
본 명세서에서, 상기 'cRANKL'는 사람, 마우스, 치킨(닭), 랫트, 돼지, 소 또는 말로 이루어진 군에서 선택된 어느 하나의 종에서 유래한 유전자이고, 바람직하게는 치킨 유래 RANKL 유전자일 수 있으며, 인위적으로 합성된 재조합 유전자 서열인 것도 가능하다. In the present specification, the 'cRANKL' is a gene derived from any one species selected from the group consisting of human, mouse, chicken, rat, pig, cow, or horse, and preferably may be a chicken-derived RANKL gene, It is also possible that it is an artificially synthesized recombinant gene sequence.
상기 재조합 벡터는 도 1의 개열지도를 갖는 것이 가능하다. It is possible for the recombinant vector to have the cleavage map shown in Figure 1.
그리고, 상기 유전자는 C 말단에 히스티딘(Histidine) 태그를 더 가지는 것일 수 있다. 상기 히스티딘 태그는 1개 내지 14개의 히스티딘이 연속된 서열일 수 있고, 6개 내지 14개의 히스티딘인 것도 가능하다. 이러한 히스티딘 태그는 RANKL 단백질을 고도로 정제하기에 바람직하다. In addition, the gene may further have a histidine tag at the C terminus. The histidine tag may be a continuous sequence of 1 to 14 histidines, and may also be a sequence of 6 to 14 histidines. This histidine tag is desirable for highly purifying RANKL protein.
본 발명은 상기 재조합 벡터로 형질 도입된 재조합 유산균을 제공할 수 있다.The present invention can provide recombinant lactic acid bacteria transformed with the above recombinant vector.
상기 재조합 벡터로 형질도입된 재조합 유산균은 락토바실러스 아시도필루스, 락토바실러스 카제이, 락토바실러스 가세리, 락토바실러스 델부루키, 불가리쿠스, 락토바실러스 헬베티커스, 락토바실러스 페멘텀, 락토바실러스 파라카세이, 락토바실러스 플란타룸, 락토바실러스 루테리, 락토바실러스 람노서스, 락토바실러스The recombinant lactic acid bacteria transfected with the recombinant vector are Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus gasseri, Lactobacillus delbruchii, Bulgaricus, Lactobacillus helveticus, Lactobacillus fementum, and Lactobacillus paracasei. , Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus
갈리바리우스, 락토코쿠스 락티스, 엔테코커스 페시움, 엔테로코커스 패칼리스, 비피도박테리움 비피덤, 비피도 박테리움 브레브, 비티도박테리움 롱검, 비피도박테이움 애니말리스 및 락티스로 이루어진 군에서 선택된 어느하나일 수 있다. 보다 상세하게는 락토코쿠스 락티스일 수 있으며, 이에 한정되지는 않는다.Gallibarius, Lactococcus lactis, Enterococcus faecium, Enterococcus faecalis, Bifidobacterium bifidum, Bifidobacterium breve, Vitidobacterium longum, Bifidobacterium animalis and Lactis. It may be any one selected from the group. More specifically, it may be Lactococcus lactis, but is not limited thereto.
상기 재조합 유산균은 소장 M 세포 분화 인자인 RANKL을 생산할 수 있다.The recombinant lactic acid bacteria can produce RANKL, a small intestine M cell differentiation factor.
보다 상세하게는 소장에 유입된 항원이 체 내의 수지상세포를 만나기 위해서는 소장 상피세포를 통과해야 하는 데, 소장 점막층에는 Peyer's patch와 같은 점막면역기구(GALT: Gut-Associated Lymphoid Tissue)가 존재하고 이러한 Peyer's patch 상부에서 M세포와 같이 특별히 분화된 세포들이 소장 내 항원을 흡수하여 하부의 면역기구에 전달하는 점막면역반응을 유도한다. 이때 외부 항원을 통과시켜주는 역할을 하는 세포가 M 세포이며, RANKL은 이러한 Peyer's patch 발달 및 M세포 분화를 유도하는 분화인자이다.More specifically, in order for antigens introduced into the small intestine to meet dendritic cells in the body, they must pass through the small intestine epithelial cells. In the small intestine mucosal layer, mucosal immune mechanisms (GALT: Gut-Associated Lymphoid Tissue) such as Peyer's patches exist, and these Peyer's patches exist. At the top of the patch, specially differentiated cells, such as M cells, absorb antigens in the small intestine and deliver them to the immune system below, inducing a mucosal immune response. At this time, the cells that allow external antigens to pass through are M cells, and RANKL is a differentiation factor that induces Peyer's patch development and M cell differentiation.
따라서, 본 발명의 cRANKL이 포함된 재조합 유산균은 Peyer's patch 발달 및 M 세포 분화를 활성화시켜 소장 내 항원을 흡수하는 점막면역반응을 유도하는데 탁월한 효과를 나타낼 수 있다.Therefore, the recombinant lactic acid bacteria containing cRANKL of the present invention can exhibit an excellent effect in inducing a mucosal immune response that absorbs antigens in the small intestine by activating Peyer's patch development and M cell differentiation.
또한, 본 발명은 상기 cRANKL이 포함된 재조합 유산균을 유효성분으로 하는 면역증강용 보조제를 제공할 수 있으며, 상기 면역증강용 보조제는 소장 M 세포를 활성화시켜 경구 백신의 흡수 효율을 증가시키는 것을 목적으로 사용할 수 있다.In addition, the present invention can provide an adjuvant for immune enhancement containing recombinant lactic acid bacteria containing cRANKL as an active ingredient, and the adjuvant for immune enhancement is aimed at increasing the absorption efficiency of oral vaccines by activating small intestine M cells. You can use it.
본 발명의 일 실시예에 따르면, 본 발명의 재조합 유산균으로부터 얻은 세포 추출물을 도 9에 나타난 바와 같은 방법으로 대조군 양계(Chicken)와 본 발명에 따른 세포 추출물 투여군의 양계에 1주일간 경구 투여한 후 IBD 백신을 상기 대조군 및 투여군의 양계에 경구 투여하고 양계의 혈액을 수집하여 확인한 결과, 도 11과 같이 IBD 특이적인 IgG (Immunoglobulin G)의 양이 증가한 것을 확인하였다.According to one embodiment of the present invention, the cell extract obtained from the recombinant lactic acid bacteria of the present invention is orally administered to control chickens and chickens of the cell extract administration group according to the present invention for one week in the method shown in Figure 9, and then IBD. The vaccine was orally administered to poultry in the control and administration groups, and blood was collected from the poultry. As a result, it was confirmed that the amount of IBD-specific IgG (Immunoglobulin G) increased, as shown in Figure 11.
상기 결과로부터 cRANKL이 포함된 재조합 유산균 및 이로부터 얻은 세포 추출물은 Chicken 경구 백신의 보조제로서 효과적이라는 것을 확인할 수 있었다.From the above results, it was confirmed that the recombinant lactic acid bacteria containing cRANKL and the cell extract obtained therefrom were effective as adjuvants for Chicken oral vaccine.
이와 함께, 본 발명의 일 구현예는 상기한 재조합 유산균을 배양한 배양물로부터 얻은 세포 추출물이다. 상기 세포 추출물은 상기 배양물을 원심분리하여 배양상층액과 균체로 분리하고, 상기 분리한 균체를 파쇄한 후 원심분리해서 얻은 세포질 단백질이 용해된 상층액인 것일 수 있다. 이러한 세포 추출물은 세포 분쇄 후 세포에서 나온 내용물들(예를 들어, nucleic acids, protein, lipid 등 cell의 성분들)을 포함할 수 있다. In addition, one embodiment of the present invention is a cell extract obtained from a culture of the above-mentioned recombinant lactic acid bacteria. The cell extract may be a supernatant in which cytoplasmic proteins are dissolved, obtained by centrifuging the culture, separating the culture supernatant and bacterial cells, disrupting the separated bacterial cells, and then centrifuging. These cell extracts may contain contents (e.g., cell components such as nucleic acids, proteins, lipids, etc.) released from cells after cell crushing.
이러한 본 발명은 재조합 유산균을 살아있는 유전자변형생물체(LMO) 상태로 사용하지 않고, 재조합 유산균을 배양한 배양물로부터 얻은 세포 추출물 형태로 이용할 수 있기 때문에, 자연생태계를 파괴하거나 환경 오염에 대한 우려가 없고, 반드시 정제 과정을 거쳐야 하는 것도 아니라는 장점이 있다. Since the present invention does not use recombinant lactic acid bacteria in the form of a living genetically modified organism (LMO), but can be used in the form of a cell extract obtained from a culture of recombinant lactic acid bacteria, there is no concern about destroying the natural ecosystem or environmental pollution. , it has the advantage of not necessarily having to go through a purification process.
본 발명의 다른 구현예는 상기한 세포 추출물을 유효성분으로 함유하는 경구 백신 보조제이다.Another embodiment of the present invention is an oral vaccine adjuvant containing the above-described cell extract as an active ingredient.
상기 경구 백신 보조제는 양계용으로 사용될때 가장 효과적일 수 있다. The oral vaccine adjuvant may be most effective when used in poultry farming.
본 발명의 또 다른 구현예는, 상기한 세포 추출물을 유효성분으로 함유하는 양계용 사료 첨가제이다. 본 발명에 따라 cRANKL이 포함된 재조합 유산균의 세포 추출물은, 소장 M세포의 분화 능력을 향상 시킬수 있고, 이로 인해 외부 항원에 대한 면역반응을 증가시켜서, 면역증강용 사료 첨가제로 활용이 가능하다. 특별히 양계용 사료 첨가제로 이용하는 것이 바람직하다. Another embodiment of the present invention is a feed additive for poultry containing the above-described cell extract as an active ingredient. According to the present invention, the cell extract of recombinant lactic acid bacteria containing cRANKL can improve the differentiation ability of small intestine M cells, thereby increasing the immune response to external antigens, and can be used as a feed additive for immunity enhancement. It is especially desirable to use it as a feed additive for poultry.
또한, 본 발명은 상기한 재조합 유산균 및 경구백신을 유효성분으로 포함하는 경구백신용 조성물을 제공할 수 있으며, 상기 경구백신용 조성물은 약학조성물로 사용될 수 있다. In addition, the present invention can provide a composition for an oral vaccine containing the above-described recombinant lactic acid bacteria and an oral vaccine as active ingredients, and the composition for an oral vaccine can be used as a pharmaceutical composition.
본 발명에 따른 약학조성물은 약학조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 또는 희석제를 더 포함할 수 있다.The pharmaceutical composition according to the present invention may further include an appropriate carrier, excipient, or diluent commonly used in the preparation of pharmaceutical compositions.
본 발명에서 사용가능한 담체, 부형제 또는 희석제로는, 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 또는 광물유 등을 들 수 있다.Carriers, excipients or diluents usable in the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, Examples include methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil.
본 발명에 따른 약학조성물은, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다.The pharmaceutical composition according to the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions according to conventional methods. You can.
제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 화합물은 적어도 하나 이상의 부형제, 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 수크로스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제한다.When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose ( It is prepared by mixing sucrose, lactose, and gelatin.
또한 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다.In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, syrups, etc. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. .
본 발명에 따른 약학조성물의 투여량은 환자의 나이, 성별, 체중에 따라 달라질 수 있으나, 재조합 유산균 0.05 mg/day 내지 10.0 mg/day 및 경구백신 0.4 mg/day 내지 10.0 mg/day의 양을 일일 1회 내지 일일 수회 분복 투여할 수 있다.The dosage of the pharmaceutical composition according to the present invention may vary depending on the patient's age, gender, and weight, but the daily dose is 0.05 mg/day to 10.0 mg/day for recombinant lactic acid bacteria and 0.4 mg/day to 10.0 mg/day for oral vaccine. It can be administered once or in divided doses several times a day.
또한, 이러한 투여량은 투여경로, 질병의 정도, 성별, 체중, 나이 등에 따라서 증감될 수 있다. 따라서, 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.Additionally, this dosage may be increased or decreased depending on the route of administration, severity of disease, gender, weight, age, etc. Accordingly, the above dosage does not limit the scope of the present invention in any way.
본 발명에 따른 경구백신용 조성물이 양계용으로 사용되는 경우, 상기 조성물의 투여량은 양계(Chicken)의 체중에 따라 달라질 수 있다. When the oral vaccine composition according to the present invention is used for poultry, the dosage of the composition may vary depending on the body weight of the chicken.
또한, 본 발명에 따른 약학조성물을 구성하는 경구백신은 이미 다른 의학적 용도에 처방되고 있어 안전성이 확보되어 있는 물질이다.In addition, the oral vaccine constituting the pharmaceutical composition according to the present invention is a substance whose safety is guaranteed as it is already prescribed for other medical purposes.
상기 약학조성물은 쥐, 생쥐, 가축, 인간 등의 포유동물에 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 자궁내 경막 또는 뇌혈관내 (intracerebroventricular)주사에 의해 투여될 수 있다.The pharmaceutical composition can be administered to mammals such as rats, mice, livestock, and humans through various routes. All modes of administration are contemplated, for example, oral, rectal or by intravenous, intramuscular, subcutaneous, intrathecal or intracerebroventricular injection.
본 발명은 하기의 실시예에 의하여 보다 더 잘 이해될 수 있으며, 하기의 실시예는 본 발명의 예시 목적을 위한 것이며, 첨부된 특허청구범위에 의하여 한정되는 보호범위를 제한하고자 하는 것은 아니다.The present invention can be better understood by the following examples, which are for illustrative purposes of the present invention and are not intended to limit the scope of protection defined by the appended claims.
<실시예 1 > 유전자 클로닝 및 단백질 발현 벡터 제작<Example 1> Gene cloning and protein expression vector construction
Chicken extracellular RANKL을 유산균 내에 발현시키기 위해, Extracellular form의 유전자 서열을 유산균 숙주에 적합하도록 코돈 최적화(Codon optimization) 후 USP45 분비 시그널과 Extracellular form RANKL을 연결한 서열(서열번호 2)을 합성의뢰하여 pTop Blunt V2 벡터에 삽입된 형태로 얻었으며, USP45-cRANKL 서열 말단에 NdeI과 XhoI 제한효소 서열도 포함이 되었다.In order to express chicken extracellular RANKL in lactic acid bacteria, codon optimization of the gene sequence of the extracellular form to suit the lactic acid bacteria host was performed, and then a sequence linking the USP45 secretion signal and the extracellular form RANKL (SEQ ID NO. 2) was requested to be synthesized to produce pTop Blunt. It was obtained as inserted into the V2 vector, and NdeI and XhoI restriction enzyme sequences were also included at the ends of the USP45-cRANKL sequence.
SEQ ID NO: 2: Nde1_ Usp45 _cRANKL_his6x_Xho1 (846bp)
상기 pTop Blunt V2 (USP45-cRANKL)은 E. coli의 Origin을 가지고 있는 벡터 시스템으로 E. coli를 대량생산하여 플라스미드를 확보한 후 NdeI과 XhoI 제한효소로 잘라 USP45-cRANKL 크기의 DNA 절편을 Gel elution하여 확보하였다. pILPtuf 벡터(pIL252 계열로부터 제작된 벡터)도 제한효소 NdeI과 XhoI으로 잘라 USP45-cRANKL 절편과 4 ℃에서 하룻밤동안 연결시킨 Cassette를 도 1의 A와 같은 지도로 제작하였다. 도 1은 본 발명의 일 실시예에 따라 USP45 분비 시그널과 Chicken 유래 RANKL 유전자를 포함하여 제작된 재조합 벡터 지도(A) 및 USP45 분비 시그널을 포함하지 않고 제작된 재조합 벡터 지도(B)이다.The pTop Blunt V2 (USP45-cRANKL) is a vector system that has the origin of E. coli . After mass producing E. coli and securing the plasmid, it was cut with NdeI and XhoI restriction enzymes and the DNA fragment of the size of USP45-cRANKL was gel eluted. It was secured. The pILPtuf vector (vector constructed from the pIL252 series) was also cut with restriction enzymes NdeI and Figure 1 shows a recombinant vector map (A) produced including the USP45 secretion signal and the Chicken-derived RANKL gene and a recombinant vector map (B) produced without the USP45 secretion signal according to an embodiment of the present invention.
연결된 벡터를 펠렛 페인팅(Pellet painting)을 진행 후 L. lactis IL1403 competent cells에 전기천공법(Electroporation)을 2.5 kV, 10 μF, 300 Ω로 진행하여 재조합 유산균 균주를 제조하였다.After pellet painting the linked vector, electroporation was performed on L. lactis IL1403 competent cells at 2.5 kV, 10 μF, and 300 Ω to prepare a recombinant lactic acid bacteria strain.
상기 방법에 따라 제작된 재조합 균주는 pILPtuf 벡터 시스템에서 USP45 분비 시그널(Secretion signal)이 도입 되었고, 세포내에서만 발현되는 특징을 나타내었으며, 유산균 밖으로 분비되지는 않았다. The recombinant strain produced according to the above method introduced the USP45 secretion signal in the pILPtuf vector system, showed the characteristic of being expressed only within the cell, and was not secreted outside the lactic acid bacteria.
이와 비교하여, 도 1의 B와 같이 USP45 분비 시그널 없이 Extracellular form RANKL을 연결한 서열(서열번호 3)을 이용한 경우에는 벡터가 만들어지지 않았고, 유산균에 transformation 시켰을 때 mutation이 발생하였다.In comparison, when a sequence (SEQ ID NO: 3) linking the extracellular form RANKL without the USP45 secretion signal was used, as shown in Figure 1B, the vector was not created, and mutations occurred when transformed into lactic acid bacteria.
SEQ ID NO: 3: Nde1_cRANKL_his6x_Xho1 (753bp)
이에 따르면, 치킨으로부터 유래된 cRANKL의 발현을 위해서는, 신호 단백질로 Usp45 분비 시그널이 필수적임을 알 수 있었다. According to this, it was found that for the expression of cRANKL derived from chicken, the Usp45 secretion signal as a signaling protein is essential.
<실시예 2> 재조합 유산균의 cRANKL 발현 확인<Example 2> Confirmation of cRANKL expression in recombinant lactic acid bacteria
상기 실시예 1의 형질전환된 L. lactis 유산균에서 cRANKL의 발현과 분비를 웨스턴 블롯(western blot)을 이용하여 검증한 후 선발된 L. lactis를 M17G(M17, 5% Glucose) 배지에서 배양하여 cRANKL의 발현 여부를 확인하였다After verifying the expression and secretion of cRANKL in the transformed L. lactis lactic acid bacteria of Example 1 using western blot, the selected L. lactis was cultured in M17G (M17, 5% Glucose) medium to produce cRANKL. The expression of
30℃에서 10시간 배양한 형질전환된 L. lactis 50ml을 4,000 rpm에서 원심분리하여 배양상층액과 균체(cell pellet)로 분리하였다. 균체를 삼차수로 2번 Washing 후, 1X PBS와 유리비트를 첨가하고 샘플파쇄기를 이용하여 39초 동안 균체를 파쇄하였다. 그 후 13,000rpm으로 원심분리하여 세포질 단백질이 용해된 상층액(세포 추출물이 포함되어 있음)을 회수하였고, 0.2 um 필터로 필터링한 후 웨스턴 블롯으로 단백질 발현을 확인하였다. 50ml of transformed L. lactis cultured at 30°C for 10 hours was centrifuged at 4,000 rpm and separated into culture supernatant and cell pellet. After washing the cells twice with tertiary water, 1X PBS and glass bits were added, and the cells were disrupted for 39 seconds using a sample crusher. Afterwards, the supernatant (containing cell extract) in which cytosolic proteins were dissolved was recovered by centrifugation at 13,000 rpm, filtered with a 0.2 um filter, and protein expression was confirmed by Western blot.
또한, 배양상층액은 TCA(trichloroacetic acid)로 단백질을 농축시켜서 확인하였다. 12 ml 배양상층액은 TCA (trichloroacetic acid)로 단백질을 침전 시킨 후 차가운 아세톤을 첨가하여 혼합한 후 세척하고 다시 원심분리한 후 상층액을 제거하였다. 그 후 남은 단백질 침전물은 Tris-HCl 200 ul에 녹여 웨스턴 블롯을 진행하여 단백질 발현을 확인하였다.In addition, the culture supernatant was confirmed by concentrating the protein with TCA (trichloroacetic acid). 12 ml of culture supernatant was precipitated with TCA (trichloroacetic acid), mixed with cold acetone, washed, centrifuged again, and the supernatant was removed. Afterwards, the remaining protein precipitate was dissolved in 200 ul of Tris-HCl and Western blot was performed to confirm protein expression.
그 결과는 도 2에 나타난 바와 같다. 도 2는 본 발명의 일 실시예에 따라 상기 균체를 파쇄하여 얻은 세포질 단백질이 용해된 상층액의 cRANKL 단백질을 확인한 Western blot 결과이다(여기서, Lane 1: cRANKL이 포함된 재조합 유산균(L. lactis (pILPtuf.cRANKL))의 세포 추출물, Lane 2: 야생형 유산균(L. lactis IL1403)의 세포 추출물. Lane 3-5: 상용 His-tagged Calmodulin (18 kDa) 1.5ug, 1ug, 0.5ug.).The results are as shown in Figure 2. Figure 2 is a Western blot result confirming the cRANKL protein in the supernatant in which cytosolic proteins obtained by disrupting the bacterial cells according to an embodiment of the present invention were dissolved (here, Lane 1: recombinant lactic acid bacteria ( L. lactis ) containing cRANKL Cell extract of pILPtuf.cRANKL), Lane 2: Cell extract of wild-type lactic acid bacteria ( L. lactis IL1403). Lane 3-5: Commercial His-tagged Calmodulin (18 kDa) 1.5ug, 1ug, 0.5ug.).
도 2에 나타난 바와 같이 형질전환된 L. lactis의 세포 추출물에서 His-tag 특이적 밴드를 확인할 수 있었다. As shown in Figure 2, a His-tag specific band was confirmed in the cell extract of transformed L. lactis .
이와 비교하여, 배양상층액에서는 USP45 분비 시그널이 연결되었지만 His-tag 특이적 밴드가 확인되지 않았다. In comparison, in the culture supernatant, a USP45 secretion signal was connected, but a His-tag specific band was not identified.
또한, Usp45-sRANKL 단백질은 서열번호 1의 아미노산 서열 C 말단에 His-tag(Histidine 6개)를 포함하는 것으로 확인되었다.In addition, the Usp45-sRANKL protein was confirmed to contain a His-tag (6 Histidines) at the C-terminus of the amino acid sequence of SEQ ID NO: 1.
<실시예 3> 재조합 유산균의 생리활성 특징 분석 및 단백질 생산량 확인<Example 3> Analysis of physiological activity characteristics of recombinant lactic acid bacteria and confirmation of protein production
상기 실시예 2에서 형질전환된 L. lactis 유산균을 배양한 후 야생형과 비교하여 균체 성장 능력을 비교 분석하였다.After culturing the L. lactis lactic acid bacteria transformed in Example 2, the growth ability of the bacterial cells was compared and analyzed compared to the wild type.
3-1. 재조합 3-1. recombination L. lactisL. lactis 유산균 균주의 성장률 확인 Check the growth rate of lactic acid bacteria strains
상기 실시예 2에서 형질전환된 L. lactis 유산균을 야생형 유산균과 비교하여 생리적 특성 변화를 확인하기 위해 시간에 따른 각각의 균주 성장률을 측정하였다.The L. lactis lactic acid bacteria transformed in Example 2 were compared with wild-type lactic acid bacteria and the growth rate of each strain over time was measured to confirm changes in physiological characteristics.
야생형과 형질전환된 L. lactis 유산균을 30℃에서 하룻밤 동안 배양하여 OD 600nm 값의 동일한 성장 수준으로 맞춘 후, 50ml의 M17G(M17 broth + 5% glucose) 새로운 배지에 500ul씩 접종 후, 1시간 간격으로 OD 600nm 값을 측정하였다.Wild type and transformed L. lactis lactic acid bacteria were cultured overnight at 30°C to achieve the same growth level of OD 600nm, then inoculated in 500ul each into 50ml of M17G (M17 broth + 5% glucose) new medium, at 1-hour intervals. The OD 600nm value was measured.
도 3은 야생형(Wild type) 유산균과 본 발명의 일 실시예에 따른 재조합 유산균의 성장율을 확인한 결과이다.Figure 3 shows the results of confirming the growth rate of wild type lactic acid bacteria and recombinant lactic acid bacteria according to an embodiment of the present invention.
도 3에 나타난 바와 같이 형질전환체가 야생형과 비교하여 지수기(Log phase)로 들어가는 시점이 다소 지연되는 것이 확인되었으나, 최종 24시간 동안 측정결과 큰 차이가 나타나지 않았다.As shown in Figure 3, it was confirmed that the transformant entered the exponential phase (log phase) somewhat delayed compared to the wild type, but there was no significant difference in the measurement results for the final 24 hours.
상기 결과로부터 본 발명에 따라 형질전환된 L. lactis 유산균 균주는 정상적인 유산균의 생리적 특성을 유지하고 있었다.From the above results, the L. lactis lactic acid bacteria strain transformed according to the present invention maintained the physiological characteristics of normal lactic acid bacteria.
3-2. 재조합 3-2. recombination L. lactisL. lactis 유산균의 단백질 생산 능력 확인 Confirmation of protein production ability of lactic acid bacteria
상기 실시예 2에서 형질전환된 L. lactis의 cRANKL 생산효율을 검증하기 위해, 형질전환된 L. lactis과 야생형 균주를 10시간 동안 배양 후, His-tag 특이적인 항체를 이용하여 세포내 생산된 cRANKL 단백질의 함량을 상용 His-tagged Calmodulin의 standard curve를 활용하여 측정하였다.To verify the cRANKL production efficiency of L. lactis transformed in Example 2, the transformed L. lactis and the wild-type strain were cultured for 10 hours, and intracellularly produced cRANKL was cultured using a His-tag specific antibody. Protein content was measured using the standard curve of commercially available His-tagged Calmodulin.
도 4는 본 발명의 일 실시예에 따른 재조합 유산균의 RANKL 생산량을 측정하기 위한 상용 Calmodulin의 standard curve이다.Figure 4 is a standard curve of commercial Calmodulin for measuring RANKL production of recombinant lactic acid bacteria according to an embodiment of the present invention.
도 4에 나타난 standard curve를 활용하여 측정한 결과, cRANKL의 세포 내 생산량은 2.2 ug/ml 이었다.As a result of measurement using the standard curve shown in Figure 4, the intracellular production amount of cRANKL was 2.2 ug/ml.
<실시예 4> <Example 4> In vitroIn vitro 에서 재조합 유산균에 의해 생산된 cRANKL의 기능성 평가Functional evaluation of cRANKL produced by recombinant lactic acid bacteria
재조합 유산균에서 생산된 cRANKL 단백질의 기능성을 확인하기 위해, 마우스 대식세포인 RAW 264.7 세포주에 본 발명에 따라 cRANKL이 포함된 재조합 유산균 세포 추출물을 처리했을 때 파골세포 유사 세포(Osteoclast-like cell)로 분화하는지를 확인하였다.In order to confirm the functionality of the cRANKL protein produced from recombinant lactic acid bacteria, when the mouse macrophage RAW 264.7 cell line was treated with the recombinant lactic acid bacteria cell extract containing cRANKL according to the present invention, it differentiated into osteoclast-like cells. I confirmed that it was working.
도 5는 본 발명의 일 실시예에 따른 재조합 유산균에서 생성된 cRANKL의 생물학적 활성을 확인하기 위해, in vitro에서 RAW 264.7 세포에 cRANKL이 포함된 재조합 유산균 세포 추출물을 처리한 후 RAW 264.7 세포가 Osteoclast-like 세포로 분화하는지를 분석(Cell differentiation assay)한 과정을 나타낸 모식도이다.Figure 5 shows that in order to confirm the biological activity of cRANKL produced from recombinant lactic acid bacteria according to an embodiment of the present invention, RAW 264.7 cells were treated with a recombinant lactic acid bacteria cell extract containing cRANKL in vitro, and then RAW 264.7 cells showed Osteoclast- This is a schematic diagram showing the process of analyzing whether cells differentiate into like cells (Cell differentiation assay).
도 5에 나타난 바와 같이, RAW 264.7 세포를 DMEM (Dulbecco's Modified Eagle's Medium) 배지(10% FBS, 1% P/S)에서 2X106 수준으로 접종하고, 4시간 후 M-CSF (Macrophage colony-stimulating factor)와 cRANKL이 포함된 재조합 유산균 세포 추출물을 첨가하였다. 3일 후, 배지와 첨가물을 새로 갈아주었고, 6일 후 RAW 264.7 세포의 RNA를 추출하여 cDNA로 합성하고 RT-PCR을 진행하였다.As shown in Figure 5, RAW 264.7 cells were inoculated at a level of 2 ) and recombinant lactic acid bacteria cell extract containing cRANKL were added. After 3 days, the medium and additives were replaced, and after 6 days, RNA from RAW 264.7 cells was extracted, synthesized into cDNA, and RT-PCR was performed.
도 6은 본 발명의 일 실시예에 따른 재조합 유산균에서 생성된 cRANKL의 생물학적 활성을 확인하기 위해, 상기 도 5의 모식도에서와 같이 cRANKL이 포함된 재조합 유산균 세포 추출물을 처리한 RAW 264.7 세포에서 추출한 RNA로 Real-time (RT) PCR을 진행하여 Osteoclast-like 세포 분화와 관련된 유전자인 Tartrate-resistant acid phosphatase (TRAP)의 발현 정도를 분석한 결과이다. Figure 6 shows RNA extracted from RAW 264.7 cells treated with recombinant lactic acid bacteria cell extract containing cRANKL as shown in the schematic diagram of Figure 5 to confirm the biological activity of cRANKL produced from recombinant lactic acid bacteria according to an embodiment of the present invention. This is the result of analyzing the expression level of tartrate-resistant acid phosphatase (TRAP), a gene related to osteoclast-like cell differentiation, by performing real-time (RT) PCR.
본 실험에서 통계분석은 one-way analysis of variance (ANOVA)을 사용하였고, 사후 검정은 Tukey’s post-hoc test를 사용하였으며, 그래프 상단의 동일한 문자는 평균값이 통계적으로 유의적인 차이가 없음을 의미한다.In this experiment, one-way analysis of variance (ANOVA) was used for statistical analysis, and Tukey's post-hoc test was used for post-hoc testing. The same letters at the top of the graph mean that there is no statistically significant difference in the mean values.
도 6에 나타난 바와 같이, 상용으로 판매되고 있는 mouse RANKL(60 ng/ml 수준)을 첨가하여 비교했을 시, 상용 mouse RANKL(positive)과 cRANKL이 포함된 재조합 유산균 세포 추출물 그룹 모두 PBS, 야생형 유산균 세포 추출물을 급여한 그룹들보다, 파골세포형성과 관련된 TRAP 유전자의 발현량이 유의적으로 증가한 것을 확인할 수 있었다. As shown in Figure 6, when compared with the addition of commercially available mouse RANKL (level of 60 ng/ml), both commercial mouse RANKL (positive) and cRANKL-containing recombinant lactic acid bacteria cell extract groups were used in PBS and wild-type lactic acid bacteria cells. It was confirmed that the expression level of TRAP gene related to osteoclast formation was significantly increased compared to the group fed the extract.
상기 결과로부터 재조합 유산균체에서 생성된 cRANKL이 생물학적 활성이 존재함을 확인할 수 있었다.From the above results, it was confirmed that cRANKL produced from recombinant lactic acid bacteria had biological activity.
<실시예 5> <Example 5> In vivoIn vivo 에서 재조합 유산에 의해 생산된 cRANKL의 점막 면역 증진 효과 확인Confirmed the mucosal immunity-promoting effect of cRANKL produced by recombinant lactic acid.
cRANKL이 포함된 재조합 유산균의 세포 추출물에 의해 Mouse 소장 내 M 세포 수가 증가 했는지를 검정하기 위해 4주령 BALB/c mouse를 이용하여 도 6과 같은 과정으로 in vivo 급여 실험을 수행하였다. To test whether the number of M cells in the small intestine of mice was increased by cell extracts of recombinant lactic acid bacteria containing cRANKL , an in vivo feeding experiment was performed using 4-week-old BALB/c mice in the same manner as shown in Figure 6.
도 7은 본 발명의 일 실시예에 따라 4주령 female BLAB/c mouse model에서 cRANKL이 포함된 재조합 유산균 세포 추출물이 점막면역증진 효과를 확인하기 위해 수행된 in vivo 분석 모식도이다.Figure 7 is a schematic diagram of an in vivo analysis performed to confirm the effect of recombinant lactic acid bacteria cell extract containing cRANKL in enhancing mucosal immunity in a 4-week-old female BLAB/c mouse model according to an embodiment of the present invention.
즉, 7일간 Mouse에게 PBS, 야생형 유산균 (균수) 세포 추출물 혹은 cRANKL이 포함된 재조합 유산균 (균수) 세포 추출물을 매일 한번씩 경구 투여하고, 안락사 후 회장 부분을 채취하여 RNA를 추출하였다. RNA는 cDNA로 합성 후 M 세포의 분화 능력을 GP2 mRNA 발현량으로 확인하였다.That is, mice were orally administered PBS, wild-type lactic acid bacteria cell extract, or recombinant lactic acid bacteria cell extract containing cRANKL once a day for 7 days, and after euthanasia, the ileum was collected and RNA was extracted. After RNA was synthesized into cDNA, the differentiation ability of M cells was confirmed by the expression level of GP2 mRNA.
도 8은 도 7의 모식도에서와 같이 야생형 유산균 세포 추출물과 본 발명의 일 실시예에 따라 cRANKL이 포함된 재조합 유산균 세포 추출물이 경구 투여된 BALB/c mouse의 소장 내 M 세포 마커의 발현량을 RT-PCR로 측정한 결과이다. GP2 (Glycoprotein 2)는 M 세포의 마커이다. control(PBS, WT_CE)그룹과 비교하여 본 발명에 따른 cRANKL_CE 그룹에서, M 세포의 마커가 유의적으로 많이 발현되는지를 판단하여, M 세포의 분화 능력이 크게 향상 되었을 것으로 판단할 수 있다.Figure 8 shows the expression level of M cell markers in the small intestine of BALB/c mouse orally administered wild-type lactic acid bacteria cell extract and a recombinant lactic acid bacteria cell extract containing cRANKL according to an embodiment of the present invention, as shown in the schematic diagram of Figure 7. RT -This is a result measured by PCR. Glycoprotein 2 (GP2) is a marker for M cells. By determining whether M cell markers are expressed significantly more in the cRANKL_CE group according to the present invention compared to the control (PBS, WT_CE) group, it can be determined that the differentiation ability of M cells is greatly improved.
그 결과, 본 발명에 따라 cRANKL이 포함된 재조합 유산균 세포 추출물을 투여한 경우, 야생형 유산균 추출물보다 GP2 수치가 높아서 M 세포의 분화 능력이 크게 향상 되었음을 확인할 수 있었다. As a result, it was confirmed that when the recombinant lactic acid bacteria cell extract containing cRANKL was administered according to the present invention, the GP2 level was higher than that of the wild-type lactic acid bacteria extract, greatly improving the differentiation ability of M cells.
<실시예 6> <Example 6> In vivoIn vivo 에서 재조합 유산균에 의해 생산된 cRANKL의 점막 면역 증진 효과 확인Confirmed the mucosal immunity enhancing effect of cRANKL produced by recombinant lactic acid bacteria
본 발명에 따라 cRANKL이 포함된 재조합 유산균의 세포 추출물에 의해 Chicken 소장 내 M 세포의 분화 능력이 향상 되었는지를 검정하기 위해, ROSS 308 2일령 양계를 이용하여 도 9에 나타난 바와 같은 과정으로 in vivo 급여 실험을 수행하였다. In order to test whether the differentiation ability of M cells in the chicken intestine was improved by the cell extract of recombinant lactic acid bacteria containing cRANKL according to the present invention, ROSS 308 was fed in vivo using 2-day-old poultry chickens through the process shown in Figure 9. An experiment was performed.
도 9는 본 발명의 일 실시예에 따라 1일령 ROSS 308 양계에서 cRANKL이 포함된 재조합 유산균 세포 추출물의 점막면역증진 효과를 확인하기 위해 수행된 in vivo 분석 모식도이다.Figure 9 is a schematic diagram of an in vivo analysis performed to confirm the mucosal immunity enhancing effect of a recombinant lactic acid bacteria cell extract containing cRANKL in 1-day-old ROSS 308 chickens according to an embodiment of the present invention.
즉, 12일간 양계에 PBS, 야생형 유산균 (균수) 세포 추출물 및 본 발명에 따라 cRANKL이 포함된 재조합 유산균 (균수) 세포 추출물을 각각 매일 한번씩 경구 투여하고, 안락사 후 회장 Peyer's patch 부분을 채취하여 RNA를 추출하였다. RNA는 cDNA로 합성 후 M 세포 마커의 발현량을 ANXA5 마커로 확인하였다.That is, for 12 days, chickens were orally administered PBS, wild-type lactic acid bacteria cell extract, and recombinant lactic acid bacteria cell extract containing cRANKL according to the present invention once daily, and after euthanasia, the Peyer's patch portion of the ileum was collected and RNA was collected. Extracted. After RNA was synthesized into cDNA, the expression level of the M cell marker was confirmed using the ANXA5 marker.
도 10A는 도 9의 모식도에서와 같이 본 발명의 일 실시예에 따른 cRANKL이 포함된 재조합 유산균 세포 추출물의 경구 투여 최적 Dose를 결정하기 위해, 여러가지 양의 cRANKL이 포함된 재조합 유산균의 세포 추출물이 경구 투여된 Hy-Line Chicken의 소장 내 M 세포 마커의 발현량을 RT-PCR로 측정한 결과이다. ANXA 5 (Annexin 5)는 M 세포의 마커이다. M 세포의 마커(ANXA5)가 유의적으로 많이 발현되는지를 판단하여, M 세포의 분화 능력이 향상 되었을 것으로 판단할 수 있다.10A is a schematic diagram of FIG. 9, in order to determine the optimal dose for oral administration of a recombinant lactic acid bacteria cell extract containing cRANKL according to an embodiment of the present invention, cell extracts of recombinant lactic acid bacteria containing various amounts of cRANKL are administered orally. This is the result of measuring the expression level of M cell markers in the small intestine of administered Hy-Line Chicken by RT-PCR. ANXA 5 (Annexin 5) is a marker of M cells. By determining whether the M cell marker (ANXA5) is expressed significantly, it can be determined that the differentiation ability of M cells has improved.
도 10A에서, C는 Control(PBS 급여)이고, T1 (cRANKL 12.1 ug/day, 5.5 mL broth에서 자란 세포들의 추출물), T2 (cRANKL 36.3 ug/day, 16.5 mL broth에서 자란 세포들의 추출물), 및 T3 (cRANKL 72.6 ug/day, 33 mL broth에서 자란 세포들의 추출물)는 본 발명에 따른 cRANKL이 포함된 재조합 L. lactis의 세포 추출물을 급여한 경우를 나타낸다. 이에 따르면, T2의 경우가 M 세포 마커의 발현량이 가장 높았던 것을 확인할 수 있다.In Figure 10A, C is Control (PBS fed), T1 (cRANKL 12.1 ug/day, extract of cells grown in 5.5 mL broth), T2 (cRANKL 36.3 ug/day, extract of cells grown in 16.5 mL broth), and T3 (cRANKL 72.6 ug/day, extract of cells grown in 33 mL broth) represents the case where the cell extract of recombinant L. lactis containing cRANKL according to the present invention was fed. According to this, it can be confirmed that the expression level of M cell markers was highest in the case of T2.
도 10B는 도 9의 모식도에서와 같이 야생형 유산균 세포 추출물과 본 발명의 일 실시예에 따라 cRANKL이 포함된 재조합 유산균 세포 추출물이 경구 투여된 ROSS 308 Chicken의 소장 내 M 세포 마커의 발현량을 RT-PCR로 측정한 결과이다. ANXA 5 (Annexin 5)는 M 세포의 마커이다. control(PBS, WT_CE)그룹과 비교하여 본 발명에 따른 cRANKL 그룹에서, M 세포의 마커가 유의적으로 많이 발현되는지를 판단하여, M 세포의 분화 능력이 향상 되었을 것으로 판단할 수 있다.Figure 10B shows the expression level of M cell markers in the small intestine of ROSS 308 Chicken orally administered wild-type lactic acid bacteria cell extract and a recombinant lactic acid bacteria cell extract containing cRANKL according to an embodiment of the present invention as shown in the schematic diagram of Figure 9. RT- This is the result measured by PCR. ANXA 5 (Annexin 5) is a marker of M cells. By determining whether M cell markers are expressed significantly more in the cRANKL group according to the present invention compared to the control (PBS, WT_CE) group, it can be determined that the differentiation ability of M cells has improved.
그 결과, 본 발명에 따라 cRANKL이 포함된 재조합 유산균 세포 추출물을 투여한 경우, 야생형 유산균 세포 추출물보다 ANXA 5수치가 높아서 M 세포의 분화 능력이 크게 향상 되었음을 확인할 수 있었다. As a result, it was confirmed that when the recombinant lactic acid bacteria cell extract containing cRANKL was administered according to the present invention, the level of ANXA 5 was higher than that of the wild-type lactic acid bacteria cell extract, and the differentiation ability of M cells was greatly improved.
<실시예 7> <Example 7> In vivoIn vivo 에서 재조합 유산균에 의해 생산된 cRANKL의 경구 백신 보조제로서의 가능성 확인Confirmed the potential of cRANKL produced by recombinant lactic acid bacteria as an oral vaccine adjuvant.
본 발명에 따라 cRANKL이 포함된 재조합 유산균의 세포 추출물이 경구 백신 보조제로서의 가능성을 검정하기 위해, ROSS 308 1일령 양계를 이용하여 도 8과 같은 과정으로 in vivo 면역 실험을 수행하였다. In order to test the possibility of the cell extract of recombinant lactic acid bacteria containing cRANKL according to the present invention as an oral vaccine adjuvant, an in vivo immune experiment was performed using ROSS 308 1-day-old poultry chickens through the process shown in Figure 8.
즉, 12일간 양계에 PBS, 야생형 유산균 (균수) 세포 추출물 및 본 발명에 따라 cRANKL이 포함된 재조합 유산균 (균수) 세포 추출물을 동일한 양으로 각각 매일 한번씩 경구 투여하고, 실험 개시 14일 차에 상용 IBD 경구 백신((주)중앙백신연구소에서 구매한 포울샷® 감보로(PoulShot® Gumboro))을 경구 투여하였다. 2주간의 항체 형성기간 후 정맥 채혈을 하여 혈청을 분리하고, 항원 특이적 항체를 ELISA assay를 통해 확인하였다. That is, the same amounts of PBS, wild-type lactic acid bacteria (bacterial count) cell extract, and recombinant lactic acid bacteria (bacterial count) cell extract containing cRANKL according to the present invention were orally administered to poultry chickens once a day for 12 days, and commercial IBD was administered on the 14th day of the experiment. Oral vaccine (PoulShot® Gumboro purchased from JoongAng Vaccine Research Institute, Inc.) was administered orally. After a 2-week antibody formation period, venous blood was collected, serum was separated, and antigen-specific antibodies were confirmed through ELISA assay.
도 11은 도 9의 모식도에서와 같이 야생형 유산균 세포 추출물과 본 발명의 일 실시예에 따라 cRANKL이 포함된 재조합 유산균 세포 추출물이 경구 투여된 ROSS 308 Chicken에게 Infectious bursal disease (IBD) 백신을 경구 투여 후 항원 특이적 항체의 생산량을 비교한 결과이다.Figure 11 shows the wild-type lactic acid bacteria cell extract as shown in the schematic diagram of Figure 9 and the recombinant lactic acid bacteria cell extract containing cRANKL according to an embodiment of the present invention after oral administration of the Infectious bursal disease (IBD) vaccine to ROSS 308 Chicken. This is the result of comparing the production amount of antigen-specific antibodies.
그 결과, 도 11에 나타난 바와 같이 대조군에 비해 본 발명에 따라 cRANKL이 포함된 재조합 유산균 세포 추출물을 급여한 그룹에서 유의적으로 항원 특이적 항체(IgG)가 높게 검출 되었다.As a result, as shown in Figure 11, significantly higher antigen-specific antibodies (IgG) were detected in the group fed with the recombinant lactic acid bacteria cell extract containing cRANKL according to the present invention compared to the control group.
상기 결과로부터 cRANKL이 포함된 재조합 유산균 세포 추출물은 Chicken 경구 백신의 보조제로서 가능성이 확인되었다.From the above results, the potential of the recombinant lactic acid bacteria cell extract containing cRANKL as an adjuvant for Chicken oral vaccine was confirmed.
상기에서는 본 발명을 특정의 바람직한 실시예에 관련하여 도시하고 설명하였지만, 이하의 특허청구범위에 의해 마련되는 본 발명의 기술적 특징이나 분야를 이탈하지 않는 한도 내에서 본 발명이 다양하게 개조 및 변화될 수 있다는 것은 당업계에서 통상의 지식을 가진 자에게 명백한 것이다. Although the present invention has been shown and described in relation to specific preferred embodiments in the above, the present invention may be modified and changed in various ways without departing from the technical features or field of the present invention defined by the following claims. It is obvious to those skilled in the art that this can be done.
<110> KNU-Industry Cooperation Foundation <120> Recombinant vector comprising genes encoding RANKL protein and immunological adjuvant having the same <130> PA-2022-20 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 272 <212> PRT <213> Artificial Sequence <220> <223> USP45_cRANKL <400> 1 Met Lys Lys Lys Ile Ile Ser Ala Ile Leu Met Ser Thr Val Ile Leu 1 5 10 15 Ser Ala Ala Ala Pro Leu Ser Gly Val Tyr Ala Asp Thr Asp Pro Ser 20 25 30 Arg Ile Ser Lys Glu Asp Ala His Cys Val Arg Met Leu Phe Arg Ser 35 40 45 Gln Glu Ser Ile Gly Leu Gln Asp Thr Pro Phe Glu Asn Gln Glu Val 50 55 60 Lys Leu Met Pro Glu Ser Cys Arg Arg Met Lys Arg Ala Leu Gln Arg 65 70 75 80 Ala Val Gln Lys Glu Val Gln Arg Ile Leu Gly Lys Glu Ser Pro Arg 85 90 95 Pro Glu Lys Ala Ala Met Glu Ala Ile Gly Met Glu Leu Tyr Arg Arg 100 105 110 Asn Lys Pro Glu Lys Gln Pro Phe Ala His Leu Ile Ile Asp Asp Lys 115 120 125 Asn Ile Leu Thr Gly Thr Arg Lys Val Asn Leu Thr Ser Trp His His 130 135 140 Asn Lys Gly Gln Ala Asn Leu Ser Asn Met Thr Phe Ser Asp Gly Lys 145 150 155 160 Leu Ile Val Asn Gln Asp Gly Phe Tyr Tyr Leu Tyr Ala Asn Ile Cys 165 170 175 Phe Arg His His Glu Thr Ser Gly Asn Leu Thr Lys Arg Gly Leu Gln 180 185 190 Leu Met Val Tyr Met Thr Lys Thr Asn Leu Lys Ile Arg Arg Ser Asp 195 200 205 Val Leu Met Lys Gly Gly Ser Thr Lys Tyr Trp Ser Gly Asn Ser Glu 210 215 220 Phe His Phe Tyr Ser Val Asn Ile Gly Gly Phe Leu Lys Leu Lys Thr 225 230 235 240 Gly Asp Met Ile Ser Ile Gln Val Ser Asn Pro Leu Leu Leu Asp Ser 245 250 255 Ser Gln Glu Ala Thr Tyr Phe Gly Ala Phe Lys Val Arg Asp Leu Asp 260 265 270 <210> 2 <211> 846 <212> DNA <213> Artificial Sequence <220> <223> Nde1_Usp45_gExRANKL_his6x_Xho1 <400> 2 catatgaaga agaagatcat cagtgcaatt cttatgtcaa ccgttatttt atctgctgcc 60 gctccattgt ctggtgtgta tgctgataca gatccatcac gtatttcaaa agaagatgct 120 cattgtgttc gtatgctttt tcgttcacaa gaatcaattg gtcttcaaga tactccattt 180 gaaaatcaag aagttaaact tatgccagaa tcatgtcgtc gtatgaaacg tgctcttcaa 240 cgtgctgttc aaaaagaagt tcaacgtatt cttggtaaag aatcaccacg tccagaaaaa 300 gctgctatgg aagctattgg tatggaactt tatcgtcgta ataaaccaga aaaacaacca 360 tttgctcatc ttattattga tgataaaaat attcttactg gtactcgtaa agttaatctt 420 acttcatggc atcataataa aggtcaagct aatctttcaa atatgacttt ttcagatggt 480 aaacttattg ttaatcaaga tggtttttat tatctttatg ctaatatttg ttttcgtcat 540 catgaaactt caggtaatct tactaaacgt ggtcttcaac ttatggttta tatgactaaa 600 actaatctta aaattcgtcg ttcagatgtt cttatgaaag gtggttcaac taaatattgg 660 tcaggtaatt cagaatttca tttttattca gttaatattg gtggttttct taaacttaaa 720 actggtgata tgatttcaat tcaagtttca aatccacttc ttcttgattc atcacaagaa 780 gctacttatt ttggtgcttt taaagttcgt gatcttgatc accatcatca ccaccattga 840 ctcgag 846 <110> KNU-Industry Cooperation Foundation <120> Recombinant vector comprising genes encoding RANKL protein and immunological adjuvant having the same <130> PA-2022-20 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 272 <212> PRT <213> Artificial Sequence <220> <223> USP45_cRANKL <400> 1 Met Lys Lys Lys Ile Ile Ser Ala Ile Leu Met Ser Thr Val Ile Leu 1 5 10 15 Ser Ala Ala Ala Pro Leu Ser Gly Val Tyr Ala Asp Thr Asp Pro Ser 20 25 30 Arg Ile Ser Lys Glu Asp Ala His Cys Val Arg Met Leu Phe Arg Ser 35 40 45 Gln Glu Ser Ile Gly Leu Gln Asp Thr Pro Phe Glu Asn Gln Glu Val 50 55 60 Lys Leu Met Pro Glu Ser Cys Arg Arg Met Lys Arg Ala Leu Gln Arg 65 70 75 80 Ala Val Gln Lys Glu Val Gln Arg Ile Leu Gly Lys Glu Ser Pro Arg 85 90 95 Pro Glu Lys Ala Ala Met Glu Ala Ile Gly Met Glu Leu Tyr Arg Arg 100 105 110 Asn Lys Pro Glu Lys Gln Pro Phe Ala His Leu Ile Ile Asp Asp Lys 115 120 125 Asn Ile Leu Thr Gly Thr Arg Lys Val Asn Leu Thr Ser Trp His His 130 135 140 Asn Lys Gly Gln Ala Asn Leu Ser Asn Met Thr Phe Ser Asp Gly Lys 145 150 155 160 Leu Ile Val Asn Gln Asp Gly Phe Tyr Tyr Leu Tyr Ala Asn Ile Cys 165 170 175 Phe Arg His His Glu Thr Ser Gly Asn Leu Thr Lys Arg Gly Leu Gln 180 185 190 Leu Met Val Tyr Met Thr Lys Thr Asn Leu Lys Ile Arg Arg Ser Asp 195 200 205 Val Leu Met Lys Gly Gly Ser Thr Lys Tyr Trp Ser Gly Asn Ser Glu 210 215 220 Phe His Phe Tyr Ser Val Asn Ile Gly Gly Phe Leu Lys Leu Lys Thr 225 230 235 240 Gly Asp Met Ile Ser Ile Gln Val Ser Asn Pro Leu Leu Leu Asp Ser 245 250 255 Ser Gln Glu Ala Thr Tyr Phe Gly Ala Phe Lys Val Arg Asp Leu Asp 260 265 270 <210> 2 <211> 846 <212> DNA <213> Artificial Sequence <220> <223> Nde1_Usp45_gExRANKL_his6x_Xho1 <400> 2 catatgaaga agaagatcat cagtgcaatt cttatgtcaa ccgttatattt atctgctgcc 60 gctccattgt ctggtgtgta tgctgataca gatccatcac gtatttcaaa agaagatgct 120 cattgtgttc gtatgctttt tcgttcacaa gaatcaattg gtcttcaaga tactccattt 180 gaaaatcaag aagttaaact tatgccagaa tcatgtcgtc gtatgaaacg tgctcttcaa 240 cgtgctgttc aaaaagaagt tcaacgtatt cttggtaaag aatcaccacg tccagaaaaa 300 gctgctatgg aagctattgg tatggaactt tatcgtcgta ataaaccaga aaaacaacca 360 tttgctcatc ttattattga tgataaaaat attcttactg gtactcgtaa agttaatctt 420 acttcatggc atcataataa aggtcaagct aatctttcaa atatgacttt ttcagatggt 480 aaacttattg ttaatcaaga tggtttttat tatctttatg ctaatatttg ttttcgtcat 540 catgaaactt caggtaatct tactaaacgt ggtcttcaac ttatggttta tatgactaaa 600 actaatctta aaattcgtcg ttcagatgtt cttatgaaag gtggttcaac taaatattgg 660 tcaggtaatt cagaatttca tttttattca gttaatattg gtggttttct taaacttaaa 720 actggtgata tgatttcaat tcaagtttca aatccacttc ttcttgattc atcacaagaa 780 gctacttatt ttggtgcttt taaagttcgt gatcttgatc accatcatca ccaccattga 840 ctcgag 846
Claims (13)
A recombinant vector containing a gene encoding a cRANKL (chicken Receptor activator of nuclear factor-kB ligand) protein represented by the amino acid of SEQ ID NO: 1 and linked to the Usp45 secretion signal.
상기 재조합 벡터는 도 1의 개열지도를 갖는 것을 특징으로 하는 재조합 벡터.
According to paragraph 1,
The recombinant vector is characterized in that it has the cleavage map of Figure 1.
상기 유전자는 C 말단에 6 히스티딘(Histidine) 태그를 더 가지는 것을 특징으로 하는 재조합 벡터.
According to paragraph 1,
A recombinant vector characterized in that the gene further has 6 histidine tags at the C terminus.
A recombinant lactic acid bacteria transduced with the recombinant vector according to any one of claims 1 to 3.
상기 재조합 유산균은 소장 M 세포 분화 인자인 RANKL (Receptor activator of nuclear factor kappa-B ligand)을 생산하는 것을 특징으로 하는 재조합 유산균.
According to paragraph 4,
The recombinant lactic acid bacteria is characterized in that it produces RANKL (Receptor activator of nuclear factor kappa-B ligand), a small intestine M cell differentiation factor.
A cell extract obtained from a culture of the recombinant lactic acid bacteria according to claim 4.
상기 세포 추출물은 상기 배양물을 원심분리하여 배양상층액과 균체로 분리하고, 상기 분리한 균체를 파쇄한 후 원심분리해서 얻은 세포질 단백질이 용해된 상층액인 것을 특징으로 하는 세포 추출물.
According to clause 6,
The cell extract is a cell extract characterized in that it is a supernatant in which cytoplasmic proteins are dissolved, obtained by centrifuging the culture, separating the culture supernatant and bacterial cells, crushing the separated bacterial cells, and then centrifuging.
An adjuvant for immune enhancement containing the recombinant lactic acid bacteria according to paragraph 4 as an active ingredient.
An oral vaccine adjuvant containing the cell extract according to claim 6 as an active ingredient.
상기 경구 백신 보조제는 양계용인 것을 특징으로 하는 양계용 경구 백신 보조제.
According to clause 9,
The oral vaccine adjuvant for poultry farming is characterized in that the oral vaccine adjuvant is for poultry farming.
A feed additive for poultry containing the cell extract according to claim 6 as an active ingredient.
상기 면역증강용 보조제는 소장 M 세포를 활성화시켜 경구백신의 흡수 효율을 증가시키는 것을 특징으로 하는 면역증강용 보조제.
According to clause 8,
The adjuvant for immune enhancement is characterized in that it increases the absorption efficiency of oral vaccines by activating small intestine M cells.
A composition for oral vaccine comprising the recombinant lactic acid bacteria according to paragraph 4 and an oral vaccine as active ingredients.
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| Application Number | Priority Date | Filing Date | Title |
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| KR1020220052747A KR20230153038A (en) | 2022-04-28 | 2022-04-28 | Recombinant vector comprising genes encoding RANKL protein and immunological adjuvant and feed additive for chicken using the same |
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| KR1020220052747A KR20230153038A (en) | 2022-04-28 | 2022-04-28 | Recombinant vector comprising genes encoding RANKL protein and immunological adjuvant and feed additive for chicken using the same |
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| KR20160000949A (en) | 2014-06-25 | 2016-01-06 | 서울대학교산학협력단 | RANKL producing recombinant lactic acid bacteria and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20160000949A (en) | 2014-06-25 | 2016-01-06 | 서울대학교산학협력단 | RANKL producing recombinant lactic acid bacteria and use thereof |
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