KR20130057221A - Anti-cancer pharmaceutical composition - Google Patents
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- KR20130057221A KR20130057221A KR1020110123033A KR20110123033A KR20130057221A KR 20130057221 A KR20130057221 A KR 20130057221A KR 1020110123033 A KR1020110123033 A KR 1020110123033A KR 20110123033 A KR20110123033 A KR 20110123033A KR 20130057221 A KR20130057221 A KR 20130057221A
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- cancer
- protein
- pharmaceutical composition
- anticancer pharmaceutical
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Abstract
Description
본 발명은 유산균 유래의 단백질을 유효성분으로 포함하는 각종 암의 치료, 경감, 개선 또는 예방용 약제학적 조성물에 관한 것이다.
The present invention relates to a pharmaceutical composition for treating, alleviating, improving or preventing various cancers containing a protein derived from lactic acid bacteria as an active ingredient.
암은 전세계적으로 사망의 주요 원인이 되는 질병이다. 신규한 화학치료 요법의 출현이 암으로 진단받은 환자의 생존을 증가시키기는 했지만, 암 질병의 모든 발병 또는 중증도를 감소시키지는 못하고 있다.Cancer is a major cause of death worldwide. Although the emergence of new chemotherapy increases the survival of patients diagnosed with cancer, it does not reduce all the incidence or severity of cancer disease.
암이란 세포가 분화(differentiation) 능력을 상실하여 세포의 성장 및 증식이 조절되지 않아 과잉증식이 나타나는 세포의 집합을 말한다. 대부분의 암의 발병은 발암 유전자(oncogene)와 종양억제 유전자(tumor suppressor gene)의 변이가 5∼8 단계에 걸쳐 일어나 궁극적으로 암세포가 생성된다는 다단계 발암설(multistep carcinogenesis)로 설명되고 있다. 암을 유발하는 발암 유전자가 활성화되면 세포의 비정상적인 증식이 유도되며, 종양억제 유전자가 활성화되면 세포의 비정상적인 증식이 억제되고 세포사멸 프로그램이 작동되어 특정 세포를 사멸시킴으로써 암세포의 생성을 차단한다고 알려져 있다.Cancer refers to a set of cells in which cells lose their ability to differentiate and thus do not undergo regulated cell growth and proliferation, resulting in hyperproliferation. Most cancer outbreaks are described as multistep carcinogenesis, in which mutations in oncogenes and tumor suppressor genes occur over five to eight stages, ultimately resulting in cancer cells. It is known that activation of oncogenes causing cancer induces abnormal cell proliferation. When the tumor suppressor gene is activated, abnormal cell proliferation is inhibited and a cell apoptosis program is activated, thereby killing certain cells, thereby blocking the production of cancer cells.
현재 암 치료제로 사용되고 있는 항암제들은 암 세포만을 선택적으로 제거하지 못하고 증식이 빠른 모든 세포를 죽이기 때문에, 소화기관의 상피세포나 모근세포처럼 암세포 보다 빨리 증식하는 정상 세포에도 영향을 미친다. 따라서 대량의 항암제를 사용하면 이러한 정상세포까지 손상을 받아 구토, 탈모 등의 부작용이 발생하고, 또한 증식이 빠른 면역 담당관인 림프구까지 파괴하여, 세균이나 바이러스에 감염되기 쉽고, 암세포를 제압하는 힘이 오히려 떨어지고, 골수억제능력으로 인해 골수의 제반 기능도 약해진다.Currently, anticancer drugs used for cancer treatment do not only selectively remove cancer cells, but they also kill normal cells that proliferate faster than cancer cells, like epithelial cells or hair follicle cells of digestive organs, because they kill all cells with rapid growth. Therefore, when a large amount of anticancer drug is used, such normal cells are damaged, resulting in side effects such as vomiting and hair loss. Also, the lymphocyte, which is a rapidly growing immunocompetent, is destroyed and easily infected with bacteria or viruses. And the bone marrow suppression ability weakens the function of the bone marrow.
따라서 암의 발생 후 이의 치료뿐만 아니라, 암의 발생을 예방하기 위한 독성이 적고 효과적인 항암제의 개발이 절실히 요구되고 있다.Accordingly, there is a desperate need for the development of effective and low-toxic anticancer drugs to prevent the development of cancer as well as the treatment of cancer after the occurrence of cancer.
본 발명은 전술한 바와 같은 종래 기술의 문제점을 해결하기 위한 것으로, 본 발명의 하나의 목적은 부작용은 최소화하면서 치료 효율을 높일 수 있는 암의 예방 또는 치료에 유용한 약제학적 조성물을 제공하는 것이다. Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above-mentioned problems occurring in the prior art, and it is an object of the present invention to provide a pharmaceutical composition useful for prevention or treatment of cancer,
본 발명의 또 다른 양상은 암의 치료 보조제 또는 예방제로 이용가능한 식품 조성물을 제공하는 것이다.Another aspect of the present invention is to provide a food composition usable as a therapeutic adjuvant or preventive agent for cancer.
상술한 목적을 달성하기 위한 본 발명의 하나의 양상은 서열번호 1 또는 서열번호 3으로 표시되는 아미노산 서열로 이루어진 p14 단백질을 유효성분으로 하는 항암 약제학적 조성물에 관한 것이다. According to one aspect of the present invention for achieving the above object, The present invention relates to an anticancer pharmaceutical composition comprising p14 protein consisting of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3 as an active ingredient.
상술한 목적을 달성하기 위한 본 발명의 다른 양상은 상기 p14 단백질을 코딩하는 유전자(핵산)를 포함하는 암의 치료 또는 예방에 효과적인 약제학적 조성물에 관한 것이다.
Another aspect of the present invention to attain the above object is to provide a pharmaceutical composition which is effective for treating or preventing cancer comprising a gene (nucleic acid) encoding the p14 protein.
본 발명은 유산균 유래의 신규한 단백질을 유효성분으로 포함하는 암의 발병을 예방하거나 치료할 수 있는 약제학적 조성물을 제공한다. 본 발명의 조성물은 암의 예방 또는 치료 효과가 우수할 뿐만 아니라, 생균(probiotic) 또는 사균 유래의 단백질을 유효성분으로 하여 세포독성 및 부작용이 없고 효과 지속 시간이 길어 약제학적 조성물 또는 식품 조성물에 안전하게 적용될 수 있다.
The present invention provides a pharmaceutical composition capable of preventing or treating the incidence of cancer comprising as an active ingredient a novel protein derived from lactic acid bacteria. The composition of the present invention is excellent in prevention or treatment of cancer, and is free from cytotoxicity and side effects using proteins derived from probiotic or bacterium as an active ingredient, has a long duration of effect, and can be safely administered to pharmaceutical compositions or food compositions Can be applied.
도 1은 락토바실러스 파라카제이 균주의 배양물 중 10 내지 100 kDa 부분의 FPLC 결과를 나타내는 것이다.
도 2는 락토바실러스 파라카제이 균주의 배양물의 분획 16-19를 이용한 항암 효능 실험 결과를 나타내는 그래프이다.
도 3은 항암 활성이 확인된 분획에서의 단백질을 확인하기 위한 실버 염색결과를 나타내는 것이다.
도 4는 항암 활성이 확인된 단백질을 이용한 아미노산 동정을 통한 blast 검색결과를 나타내는 것이다.
도 5는 대장균 발현 벡터에 13 kDa, 14 kDa, 및 49 kDa 유전자를 삽입하기 위한 PCR 결과를 나타내는 사진이다.
도 6은 제한 효소반응과 T4 리가제를 이용한 13 kDa, 14 kDa, 및 49 kDa 유전자를 벡터에 클로닝한 결과이다.
도 7은 각각의 재조합 단백질이 과발현하는 콜로니를 선택하기 위해 이. 콜라이(E. coli) 콜로니를 각각 IPTG 유도 후 SDS-Page한 결과를 나타내는 사진이다.
도 8은 선별된 콜로니에서 과발현하는 IPTG 유도 조건을 확립하기 위해 13 kDa, 14 kDa, 및 49 kDa 유전자가 삽입되어 있는 대장균을 시간대별 유도시킨 결과를 나타내는 사진이다.
도 9a-b는 대용량 배양을 통한 본 발명의 p14 단백질의 분리 정제 결과 및 투석결과를 나타내는 것이다.
도 10은 재조합 단백질의 P13 및 p14 단백질의 아미노산 동정을 통한 상동성 비교 결과를 나타내는 것이다.
도 11a-b는 대장암 세포주 HT-29 및 DLD-1에서 MTT 분석법에 의해 항암 활성을 평가한 결과를 도시한 그래프이다.
도 12는 마우스 대식세포주를 이용한 본 발명의 p14 단백질의 세포독성 실험결과를 나타내는 세포 생존율 그래프이다.
도 13a-b는 마우스의 비장세포를 이용한 본 발명의 p14 단백질의 면역활성 실험결과를 나타내는 그래프이다.
도 14는 발암물질인 DMH에 의해 대장암이 유도된 F344/N 쥐에서 p14 단백질 처리 후의 사료섭취량을 도시한 그래프이다.
도 15a-b는 DMH 처리된 마우스의 결장 점막 내의 p14 단백질 처리 전후의 AC(aberrant crypt) 및 ACF(aberrant crypt foci)의 수를 나타내는 그래프이다.
도 16은 인간 대장암 세포주 DLD-1을 누드 마우스에 이식한 Xenograft 모델에서 종양세포의 성장을 모니터링한 결과로, p14 단백질을 투여한 경우 종양의 세포의 성장이 대조군에 비해 약 10 % 이상 저해되는 것을 보여주는 그래프이다
도 17은 인간 대장암 세포주 DLD-1을 이식 받은 누드 마우스에서 p14 단백질 투여 전과 p14 단백질 4회 투여 후의 종양의 크기를 사진 촬영하여 비교한 결과이다. Figure 1 shows the FPLC results of a 10-100 kDa portion of a culture of Lactobacillus paracase strains.
2 is a graph showing the results of an anticancer efficacy experiment using fractions 16-19 of a culture of a lactobacillus paracase strain.
FIG. 3 shows the results of silver staining for identifying proteins in the fractions in which anticancer activity was confirmed.
FIG. 4 shows the result of blast detection through amino acid identification using a protein having anticancer activity.
5 is a photograph showing PCR results for inserting 13 kDa, 14 kDa, and 49 kDa genes into an E. coli expression vector.
FIG. 6 shows the result of cloning 13 kDa, 14 kDa, and 49 kDa genes using a restriction enzyme reaction and T4 ligase into a vector.
FIG. 7 is a graph showing the effect of the recombinant protein on the expression of the < RTI ID = 0.0 > E. coli colonies after SDS-PAGE after IPTG induction, respectively.
FIG. 8 is a photograph showing the results of time-series induction of Escherichia coli having 13 kDa, 14 kDa, and 49 kDa genes inserted therein in order to establish IPTG induction conditions overexpressed in selected colonies.
FIGS. 9A and 9B show results of separation and purification of the p14 protein of the present invention and dialysis results through a large-capacity culture.
FIG. 10 shows the results of homology comparison of amino acids of P13 and p14 proteins of the recombinant protein.
11a-b are graphs showing the results of evaluating anticancer activity by MTT assay in colorectal cancer cell lines HT-29 and DLD-1.
12 is a graph of cell survival rate showing the cytotoxicity test results of the p14 protein of the present invention using a mouse macrophage cell line.
FIGS. 13A and 13B are graphs showing the results of immunological activity of p14 protein of the present invention using mouse spleen cells. FIG.
FIG. 14 is a graph showing feed intake after treatment with p14 protein in F344 / N rats in which colorectal cancer was induced by DMH, a carcinogen.
FIGS. 15A and 15B are graphs showing the numbers of AC (aberrant crypt) and ACF (aberrant crypt foci) before and after treatment of p14 protein in the colonic mucosa of DMH-treated mice.
FIG. 16 shows the results of monitoring the growth of tumor cells in a Xenograft model transplanted with human colon cancer cell line DLD-1 into a nude mouse. As a result, when the p14 protein was administered, the growth of tumor cells was inhibited by about 10% It is a graph showing that
FIG. 17 is a photograph showing the size of tumor after pre-administration of p14 protein and 4 times of administration of p14 protein in nude mice transplanted with human colon cancer cell line DLD-1.
본 발명에서 사용되는 경우에 "암"이라는 용어는 종양, 네오플라시아스(neoplasias), 및 악성 조직 또는 세포를 포함하는 의미이다. 상기 암은 대장암, 폐암, 소세포폐암, 위암, 간암, 혈액암, 골암, 췌장암, 피부암, 두부 또는 경부암, 피부 또는 안구내 흑색종, 자궁암, 난소암, 직장암, 항문부근암, 결장암, 유방암, 나팔관암종, 자궁내막암종, 자궁경부암, 질암, 음문암종, 호지킨병, 식도암, 소장암, 내분비선암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 요도암, 음경암, 전립선암, 만성 또는 급성 백혈병, 림프구 림프종, 방광암, 신장암, 수뇨관 암, 신장세포 암종, 신장골반 암종, CNS 종양, 1차 CNS 림프종, 척수 종양, 뇌간신경교종, 뇌하수체 선종과 같은 암 또는 이들 암들의 하나 이상의 조합을 포함한다. As used herein, the term "cancer" is meant to include tumors, neoplasias, and malignant tissues or cells. Wherein the cancer is selected from the group consisting of colon cancer, lung cancer, small cell lung cancer, gastric cancer, liver cancer, bone cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, Endometrioid cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute cancers, endometrioid carcinoma, endometrial carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, Cancer such as leukemia, lymphocytic lymphoma, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brain glioma, pituitary adenoma or combinations of one or more of these cancers do.
본 발명의 하나의 실시예는 서열번호 1 또는 서열번호 3으로 표시되는 아미노산 서열로 이루어진 항암 활성을 갖는 p14 단백질을 유효성분으로 하는 항암 약제학적 조성물에 관한 것이다. 본 발명의 p14 단백질은 약 14 kDa의 분자량을 갖는다. 본 발명의 조성물은 상기의 서열번호 1의 단백질의 기능적 동등물을 포함한다. 여기서 "기능적 동등물"이란 아미노산의 부가, 치환 또는 결실의 결과, 상기 서열번호 1로 표시되는 아미노산 서열과 70% 이상, 바람직하게는 80% 이상, 더욱 바람직하게는 90% 이상의 서열 상동성을 갖는 것으로, 서열번호 1로 표시되는 단백질과 실질적으로 동일한 생리활성을 나타내는 단백질을 말한다. "실질적으로 동일한 생리활성"이란 동물체 내에서 항암 활성을 의미한다. 본 발명의 일 구현예에 따른 상기 p14 단백질은 락토바실러스 카제이(Lactobacillus casei), 락토바실러스 파라카제이(Lactobacillus paracasei) 또는 락토바실러스 람노수스(Lactobacillus rhamnosus) 유래인 것을 특징으로 한다. 이들의 구체적인 예로는 락토바실루스 파라카제이 LPC4 균주 (Lactobacillus paracasei LPC4) (KCTC 11866BP) 등을 들 수 있으나, 반드시 이들로 제한되는 것은 아니다. 특히 상기 락토바실러스 람노수스(Lactobacillus rhamnosus) 유래의 p14 단백질은 서열번호 1의 아미노산 서열과 약 90%의 서열 상동성을 갖는다. 락토바실러스 람노수스(Lactobacillus rhamnosus) 유래의 p14 단백질은 서열번호 3으로 표시되는 아미노산 서열을 포함하고, 이러한 p14 단백질은 서열번호 4로 표시되는 유전자 서열에 의해 코딩될 수 있다. One embodiment of the present invention relates to an anticancer pharmaceutical composition comprising p14 protein having an anticancer activity consisting of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 as an active ingredient. The p14 protein of the present invention has a molecular weight of about 14 kDa. The composition of the present invention comprises a functional equivalent of the protein of SEQ ID NO: 1 above. As used herein, the term "functional equivalent" means a polypeptide having 70% or more, preferably 80% or more, and more preferably 90% or more homology with the amino acid sequence represented by SEQ ID NO: 1 as a result of addition, substitution or deletion Quot; refers to a protein that exhibits substantially the same physiological activity as the protein represented by SEQ ID NO: 1. "Substantially equivalent physiological activity" means an anticancer activity in a human. The p14 protein according to an embodiment of the present invention is characterized in that it is derived from Lactobacillus casei , Lactobacillus paracasei or Lactobacillus rhamnosus . Specific examples thereof include lactobacillus paracase LPC4 strain ( Lactobacillus paracasei LPC4) (KCTC 11866BP), but are not limited thereto. In particular, the Lactobacillus rhamnosus- derived p14 protein has about 90% sequence homology with the amino acid sequence of SEQ ID NO: 1. The p14 protein derived from Lactobacillus rhamnosus contains the amino acid sequence shown in SEQ ID NO: 3, and such p14 protein can be encoded by the gene sequence shown in SEQ ID NO: 4.
락토바실러스 카제이(Lactobacillus casei), 락토바실러스 파라카제이(Lactobacillus paracasei) 또는 락토바실러스 람노수스(Lactobacillus rhamnosus) 유래의 p14 단백질은 장 상피세포에 있는 Epidermal Growth Factor Receptor (EGFP)에서 인지하여 Akt로 신호를 전달하는 주요 신호전달 체계를 갖거나, 혹은 JNK 또는 MAPK의 신호 전달체계를 가질 것으로 추정된다. The p14 protein derived from Lactobacillus casei , Lactobacillus paracasei or Lactobacillus rhamnosus was recognized by Epidermal Growth Factor Receptor (EGFP) in intestinal epithelial cells and Akt signaling Or a signaling pathway of JNK or MAPK.
또한, 본 발명의 다른 양상은 상기 p14 단백질을 코딩하는 유전자(핵산)를 유효성분으로 하는 항암 약제학적 조성물에 관한 것이다. 이러한 유전자는 p14 단백질을 각각 코딩하는 게놈 DNA와 cDNA를 모두 포함한다. 바람직하게는, 상기 유전자는 서열번호 2 또는 서열번호 4로 표시되는 염기 서열을 포함한다. Another aspect of the present invention relates to an anticancer pharmaceutical composition comprising a gene (nucleic acid) encoding the p14 protein as an active ingredient. These genes include both genomic DNA and cDNA encoding each of the p14 protein. Preferably, the gene comprises the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4.
또한, 상기 염기 서열의 변이체가 본 발명의 범위 내에 포함될 수 있다. 구체적으로, 상기 유전자는 서열번호 2로 표시되는 염기 서열과 70% 이상, 바람직하게는 80% 이상, 더욱 바람직하게는 90% 이상의 서열 상동성을 가지는 염기 서열을 포함할 수 있다. 폴리뉴클레오티드에 대한 "서열 상동성의 %"는 두 개의 최적으로 배열된 서열과 비교 영역을 비교함으로써 확인되며, 비교 영역에서의 폴리뉴클레오티드 서열의 일부는 두 서열의 최적 배열에 대한 참고 서열(추가 또는 삭제를 포함하지 않음)에 비해 추가 또는 삭제(즉, 갭)를 포함할 수 있다.In addition, variants of the above base sequences can be included within the scope of the present invention. Specifically, the gene may include a nucleotide sequence having 70% or more, preferably 80% or more, and more preferably 90% or more sequence homology with the nucleotide sequence shown in SEQ ID NO: 2. "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >
상기 유전자(핵산)는 발현 벡터 내에 포함될 수 있고, 이러한 발현 벡터는 플라스미드 또는 바이러스성 벡터일 수 있다. The gene (nucleic acid) may be contained in an expression vector, and the expression vector may be a plasmid or a viral vector.
상기 핵산은 플라스미드 또는 바이러스 벡터와 같은 발현 벡터 내로 공지의 방법에 의해 도입시킨 후 상기 발현 벡터를 일시적 형질감염(transient transfection), 미세주사, 형질도입(transduction), 세포융합, 칼슘 포스페이트 침전법, 리포좀 매개된 형질감염(liposome-mediated transfection), DEAE 덱스트란-매개된 형질감염(DEAE Dextran- mediated transfection), 폴리브렌-매개된 형질감염(polybrene-mediated transfection), 전기침공법(electroporation), 유전자 건(gene gun) 및 세포 내로 DNA를 유입시키기 위한 다른 공지의 방법에 의해 발현형으로 표적 세포 내에 도입시킬 수 있다(Wu et al., J. Bio. Chem., 267:963-967, 1992; Wu and Wu, J. Bio. Chem., 263:14621-14624, 1988). The nucleic acid may be introduced into an expression vector such as a plasmid or a viral vector by a known method, and then the expression vector may be transiently transfected, microinjected, transduction, cell fusion, calcium phosphate precipitation, But are not limited to, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, electroporation, (Wu et al., J. Bio. Chem., 267: 963-967, 1992; Wu et < RTI ID = 0.0 > al., & and Wu, J. Bio. Chem., 263: 14621-14624, 1988).
플라스미드 발현 벡터는 사람에게 사용할 수 있는 FDA의 승인된 유전자 전달방법으로 사람 세포에 직접적으로 플라스미드 DNA를 전달하는 방법이다(Nabel, E. G., et al., Science, 249:1285-1288, 1990). 플라스미드 DNA는 바이러스 벡터와는 달리 균질하게 정제될 수 있는 장점이 있다. 본 발명에서 사용할 수 있는 플라스미드 발현 벡터로는 당업계에 공지된 포유동물 발현 플라스미드를 사용할 수 있다. 플라스미드 발현 벡터의 예들은 pRK5(유럽특허 제307,247호), pSV16B(국제특허공개 제91/08291호) 및 pVL1392(PharMingen)를 포함하나, 반드시 이들로 제한되는 것은 아니다.Plasmid expression vectors are FDA's approved gene delivery methods for human use that deliver plasmid DNA directly to human cells (Nabel, E. G., et al., Science, 249: 1285-1288, 1990). Plasmid DNA has the advantage that it can be homogeneously purified unlike viral vectors. As the plasmid expression vector that can be used in the present invention, mammalian expression plasmids known in the art can be used. Examples of plasmid expression vectors include, but are not necessarily limited to, pRK5 (European Patent No. 307,247), pSV16B (International Patent Publication No. 91/08291) and pVL1392 (PharMingen).
본 발명에 따른 핵산을 포함하는 바이러스 발현 벡터로는 레트로바이러스(retrovirus), 아데노바이러스(adenovirus), 헤르페스바이러스(herpes virus) 및 아비폭스바이러스(avipox virus)를 예로 들 수 있으나, 반드시 이들로 한정되는 것은 아니다.Examples of the virus expression vector containing the nucleic acid according to the present invention include retroviruses, adenoviruses, herpes viruses and avipox viruses, but are limited thereto It is not.
본 발명의 조성물은 유효성분인 항암 활성을 갖는 p14 단백질 이외에 제약학적으로 허용가능한 부형제, 희석제, 담체 또는 버퍼 등을 포함할 수 있다. The composition of the present invention may contain pharmaceutically acceptable excipients, diluents, carriers or buffers in addition to p14 protein having anticancer activity as an active ingredient.
본 발명에서 사용하기에 적합한 약제학적으로 허용되는 첨가제, 예컨대 완충제, 희석제, 담체, 보조제 또는 다른 부형제는 Remington's Pharmaceutical Sciences (19th ed., 1995)에 상세하게 기재되어 있다. 그 밖에 여러 가지 목적을 위해 다른 작용제가 조성물에 이용될 수 있다. 예를 들어, 완충제, 보존제, 공용매, 오일, 습윤제, 연화제, 안정화제 또는 항산화제를 이용할 수 있다. 이용할 수 있는 수용성 보존제로는 나트륨 비설페이트, 나트륨 티오설페이트, 벤즈알코늄 클로라이드, 클로로부탄올, 티메로살, 에틸 알콜, 메틸파라벤, 폴리비닐알콜, 벤질 알콜 및 페닐에틸 알콜이 포함된다. 이러한 작용제는 약 0.001 내지 약 5 중량％의 개별적인 양으로 존재할 수 있다. 이용할 수 있는 적합한 수용성 완충제는 원하는 투여 경로에 대해 미국 식약청 ("US FDA")이 인증한 탄산나트륨, 붕산나트륨, 인산나트륨, 아세트산 나트륨, 중탄산나트륨 등이다. 이러한 작용제는 약 2 내지 약 11 사이의 시스템의 pH를 유지하기에 충분한 양으로 존재할 수 있다. 따라서, 완충제는 전체 조성물의 중량 기준으로 약 5 중량％ 만큼 많을 수 있다. 또한, 염화나트륨 및 염화칼륨 등과 같은 전해질이 제형에 포함될 수 있다.Suitable pharmaceutically acceptable additives for use in the present invention, such as buffers, diluents, carriers, adjuvants or other excipients, are detailed in Remington's Pharmaceutical Sciences (19 th ed., 1995). Other agonists may be used in the composition for various other purposes. For example, buffers, preservatives, cosolvents, oils, wetting agents, softening agents, stabilizers or antioxidants can be used. Water-soluble preservatives that may be used include sodium bisulfate, sodium thiosulfate, benzalkonium chloride, chlorobutanol, thimerosal, ethyl alcohol, methyl paraben, polyvinyl alcohol, benzyl alcohol and phenylethyl alcohol. Such agents may be present in individual amounts from about 0.001 to about 5 weight percent. Suitable water-soluble buffers that may be employed are sodium carbonate, sodium borate, sodium phosphate, sodium acetate, sodium bicarbonate, etc., certified by the US Food and Drug Administration ("US FDA") for the desired route of administration. Such an agent may be present in an amount sufficient to maintain the pH of the system between about 2 and about 11. Thus, the buffering agent may be as much as about 5% by weight, based on the weight of the total composition. In addition, electrolytes such as sodium chloride and potassium chloride and the like may be included in the formulations.
본 발명의 p14 단백질을 의약품으로 사용하는 경우, 추가로 동일 또는 유사한 항암 효능을 나타내는 유효성분을 1종 이상 함유할 수 있다.When the p14 protein of the present invention is used as a medicine, it may further contain one or more active ingredients exhibiting the same or similar anticancer efficacy.
상기 p14 단백질은 임상 투여 시에 경구로 투여가 가능하며 일반적인 의약품 제제의 형태로 사용될 수 있다. 즉, 본 발명의 p14 단백질은 실제 임상 투여 시에 경구용의 여러 가지 제형으로 투여될 수 있다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함된다.The p14 protein can be orally administered at the time of clinical administration and can be used in the form of a general pharmaceutical preparation. That is, the p14 protein of the present invention can be administered in various oral dosage forms for actual clinical administration. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules and the like.
본 발명의 항암 약제학적 조성물의 제조방법은 p14 단백질 및 제약상 허용되는 부형제, 희석제, 담체, 또는 버퍼를 배합하는 것을 포함한다. 다른 측면에서, 본 발명의 조성물의 제조 방법은 p14 단백질, 1종 이상의 항암제 및 제약상 허용되는 부형제, 희석제, 담체, 또는 버퍼를 배합하는 것을 포함한다. 상기 항암제의 예들은 시스플라틴(ciplatin), 독소루비신(doxorubicin) 및 에토포사이드(etoposide) 등을 포함하나, 반드시 이들로 한정되는 것은 아니다. Methods of preparing the anti-cancer pharmaceutical compositions of the present invention include combining p14 protein and pharmaceutically acceptable excipients, diluents, carriers, or buffers. In another aspect, a method of making a composition of the invention comprises combining a p14 protein, one or more anti-cancer agents, and a pharmaceutically acceptable excipient, diluent, carrier, or buffer. Examples of the anticancer agents include, but are not limited to, cisplatin, doxorubicin, and etoposide.
본 발명의 약제학적 조성물은 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by those having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container.
본 발명의 항함 약제학적 조성물은 "약학적으로 유효한 양"의 p14 단백질을 포함할 수 있다. 여기서 "약학적으로 유효한 양"이란 암을 치료 또는 예방하기에 충분한 양을 말한다. 본 발명에 따른 p14 단백질의 약학적으로 유효한 양은 0.1 ~ 100mg/day/체중kg, 바람직하게는 12 mg/day/체중kg이다. An inventive pharmaceutical composition of the invention may comprise a "pharmaceutically effective amount" of the p14 protein. The term "pharmaceutically effective amount" as used herein refers to an amount sufficient to treat or prevent cancer. The pharmacologically effective amount of p14 protein according to the present invention is 0.1 to 100 mg / day / kg body weight, preferably 12 mg / day / kg body weight.
그러나 p14 단백질의 사용량은 투여경로, 질병의 정도, 성별, 체중, 나이 등에 따라서 증감될 수 있다. 따라서, 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.However, the amount of p14 protein to be used can be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and the like. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.
또 다른 실시예에서 본 발명의 p14 단백질을 포함하는 조성물은 암 관련 질환의 보조 치료를 위한 식품 및 음료 등에 다양하게 이용될 수 있다. 본 발명의 p14 단백질을 첨가할 수 있는 식품으로는, 예를 들어, 각종 식품류, 음료, 차, 비타민 복합제, 건강보조 식품류 등이 있다. 본 발명의 유산균으로부터 분리된 p14 단백질 자체는 독성 및 부작용이 거의 없으므로 예방 목적으로 장기간 복용시에도 안심하고 사용할 수 있다.In another embodiment, the composition comprising the p14 protein of the present invention can be used in a variety of foods and beverages for auxiliary treatment of cancer-related diseases. Foods to which the p14 protein of the present invention can be added include, for example, various foods, beverages, tea, vitamin complexes, and health supplement foods. Since the p14 protein itself isolated from the lactic acid bacteria of the present invention has little toxicity and side effects, it can be safely used for prophylactic purposes even for a long period of time.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention more specifically and that the scope of the present invention is not limited by these embodiments.
실시예Example
실시예 1. p14 단백질의 분리 동정 및 재조합 단백질의 생산Example 1. Isolation and Identification of p14 Protein and Production of Recombinant Protein
1-1. 락토바실러스 파라카제이의 배양물에서 항암활성이 있는 단백질의 분리 및 정제1-1. Isolation and purification of anticancer activity protein in the culture of Lactobacillus paracasei
락토바실러스 파라카제이 LPC4 균주(Lactobacillus paracasei)(KCTC 11866BP)를 페닐 세파로즈를 이용하여 분획한 분획물을 이용하여 단백질을 분리하였다. 락토바실러스 파라카제이 균주를 배양하여 농축한 후 농축물에서 항암활성을 가지는 단백질을 정제하기 위해서, FPLC(fast protein liquid chromatography, Sephadex 75)를 수행하였다. 구체적으로 버퍼로 0.05M의 인산염 및 0.15M 염화나트륨이 혼합된 pH 7.2의 용액을 사용하고, 유속을 0.5㎖/분으로 유지하였으며 시료양은 1㎖(2~4㎎/㎖)로 하고 칼럼은 Sephadex 75(분리능 Mr= 3,000~70,000)를 사용하여 수행하여 ultra filtration으로 확보된 3~100kDa, 10~30kDa의 분획에서 항암활성을 가지는 단백질 성분을 수득할 수 있었다(도 1 참조).
Lactobacillus paracase LPC4 strain ( Lactobacillus paracasei) (using a fraction fraction using the KCTC 11866BP) phenyl Sepharose was isolated protein. FPLC (fast protein liquid chromatography, Sephadex 75) was performed to purify proteins having anticancer activity in the concentrate after culturing and concentrating the Lactobacillus paracase strain. Specifically, a pH 7.2 solution containing 0.05 M phosphate and 0.15 M sodium chloride as a buffer was used, the flow rate was maintained at 0.5 ml / min, the volume of the solution was 1 ml (2-4 mg / ml), the column was Sephadex 75 (Resolution: Mr = 3,000 to 70,000), thereby obtaining a protein component having anticancer activity in a fraction of 3 to 100 kDa and 10 to 30 kDa obtained by ultrafiltration (see FIG. 1).
1-2. 항암 활성 분획의 확인 및 아미노산 동정1-2. Identification of anticancer activity fractions and identification of amino acids
16~19번 분획물을 이용하여 위암세포(AGS), 폐암세포(A549), 유방암 세포(MCF-7), 난소암 세포(SKOV-3) 및 대장암 세포(LoVo)를 24웰-플레이트에 900㎕씩 첨가하고 24시간 동안 배양(37℃, 5% CO2)하였다. 배양 후 상기 분리 정제된 분획물들을 100㎕씩 첨가하고 24시간 동안 추가 배양(37℃, 5% CO2)하였다. 배양 후, MTT(0.5㎎/㎖) 용액을 100㎕씩 추가 첨가하여 4시간 동안 다시 배양(37℃, 5% CO2)하였다. 이어서 상등액을 제거하고 300㎕의 DMSO(dimethyl sulfoxide)를 첨가하여 생성된 포르마잔(formazan)을 용해시킨 다음, ELISA 리더를 이용하여 540nm에서 흡광도를 측정하였다. 이러한 흡광도 측정 결과를 바탕으로 암세포 생육 억제활성을 하기 수식을 이용하여 억제 활성(inhibition ratio)으로 산출하여 그 결과를 도 2에 그래프로 도시하였다. 각각의 분획은 FPLC (Sephadex 75) 0.5ml/min, 3ml/분획으로 회수하였다.(AGS), lung cancer cells (A549), breast cancer cells (MCF-7), ovarian cancer cells (SKOV-3) and colorectal cancer cells (LoVo) were fractionated on a 24 well plate Mu] l and cultured for 24 hours (37 [deg.] C, 5% CO 2 ). After the culture, 100 쨉 l of the separated and purified fractions were added and further cultured (37 캜, 5% CO 2 ) for 24 hours. After the cultivation, 100 쨉 l of a solution of MTT (0.5 ㎎ / ml) was further added, and the cells were further cultured (37 캜, 5% CO 2 ) for 4 hours. Subsequently, the supernatant was removed and 300 mu l of DMSO (dimethyl sulfoxide) was added to dissolve the generated formazan, and the absorbance was measured at 540 nm using an ELISA reader. Based on the absorbance measurement results, the inhibitory activity of the cancer cell growth inhibitory activity was calculated using the following formula. The results are shown in the graph of FIG. Each fraction was recovered with FPLC (Sephadex 75) 0.5 ml / min, 3 ml / fraction.
억제 활성(Inhibition ratio(%)) = (1-T/C)ⅹ100Inhibition ratio (%) = (1-T / C) x 100
(T : 시료 첨가군의 흡광도, C : 무첨가군의 흡광도)(T: absorbance of sample addition group, C: absorbance of no addition group)
도 2를 통해서 확인되는 바와 같이, 3~100kDa 분획에서 분리된 단백질의 항암활성을 조사한 결과, 대조군에서는 암세포에 대한 세포독성은 나타내지 않았지만, 10~30kDa 분획에서 분리된 단백질의 항암활성을 조사한 결과, 농축액 중 16 내지 19번 분획이 항암 활성을 지니고 있는 물질임을 확인하였다.
As shown in FIG. 2, the anticancer activity of the protein isolated from the 3-100 kDa fraction was examined. As a result, the anti-cancer activity of the protein isolated from the 10-30 kDa fraction was examined, It was confirmed that
1-3. p14 항암 단백질의 동정1-3. Identification of p14 anticancer protein
16~19번 분획물을 이용하여 실험을 수행하였는데 실버 염색 및 코마시 블루 250 염색 모두 p14 및 p44에서 단백질이 분리되어 이를 이용하여 아미노산 동정 및 물질 규명을 수행하였다(도 3 참조). 염색에 의해서 분리된 단백질 피크를 액상 크로마토그래피/질량 스펙트로미터를 사용하여 분석한 결과 p49, p14, P13의 3 종류의 단백질이 동정되었다. 49kDa 단백질의 경우 세포벽-관련 히드롤라제(hydrolase)로 95% 이상의 유사성을 보였으며, p14 단백질은 아직 기능은 밝혀지지 않은 미지의 단백질이며 이들과도 95% 이상의 유사성을 보였다(도 4 참조).
The experiment was carried out using
1-4.1-4. 재조합 단백질의 확보Securing recombinant proteins
가. 제한효소 절단부위가 포함된 프라이머 디자인 및 제작end. Primer design and production with restriction enzyme cleavage site
13 kDa, 14 kDa과 49 kDa 유전자를 대장균 발현 벡터인 pRSET A벡터 (Invitrogen)에 클로닝 하기 위해 도 1과 같이 BamHI과 XhoI의 염기서열이 포함된프라이머를 디자인하여 제작하였다. 돌연변이되어 제한효소를 인식하지 못할 것을 대비해 염기 한 개 내지 두 개를 제한효소 인식부위와 인접하여 추가해서 디자인하였다. 리딩 프레임을 고려하여 프라이머를 디자인한 후 pRSET A 벡터에 클로닝하였다.
To clone the 13 kDa, 14 kDa and 49 kDa genes into the E. coli expression vector pRSET A vector (Invitrogen) BamHI and A primer containing the base sequence of XhoI was designed and produced. One or two bases were designed to be adjacent to the restriction enzyme recognition site in order to prevent mutation and recognition of the restriction enzyme. The primer was designed considering the leading frame and cloned into the pRSET A vector.
나. 대장균 발현 벡터에 각각의 CDS를 삽입하기 위한 PCR 수행 (도 5)I. PCR for insertion of each CDS into an E. coli expression vector (FIG. 5)
제작된 프라이머를 이용하여 각각 55℃의 어닐링 온도에서 CDS들의 PCR 산물을 확인하였다. 반응용액에는 주형 3 ㎕ (10 ng/㎕), 프라이머(10 pM), MgCl2 (25mM), dNTP(2.5mM), Taq 폴리메라제(5U/㎕), 10×버퍼가 포함되었고, 반응조건은 초기변성 (95˚C) 반응을 2분 동안 진행시킨 후, 변성반응 (95˚C) 20초, 결합 반응 (55˚C) 35초, 연장반응 (72˚C), 20초로 30회 반복하여 수행하였다. 락토바실러스 카제이(Lactobacillus casei)의 DNA를 주형으로 이용하고, PCR에 의한 돌연변이를 고려하여 high fidelity polymerase Taq invitrogen을 이용여 PCR을 수행하였다. 각각의 PCR 산물은 3종의 유전자 크기와 일치하는 각각 369, 372, 1494 bp임을 확인할 수 있었다.
PCR products of CDS were identified at annealing temperature of 55 ℃ using primers. The reaction solution contained 3 μl (10 ng / μl) of template, 10 pM of primer, 25 mM of MgCl 2 , 2.5 mM of dNTP, 5 μl of Taq Polymerase and 10 × buffer, (95 ° C) reaction for 2 minutes and 30 cycles of denaturation (95 ° C) for 20 seconds, binding reaction (55 ° C) for 35 seconds, extension reaction (72 ° C) and 20 seconds Lt; / RTI > PCR was performed using the DNA of Lactobacillus casei as a template and high fidelity polymerase Taq invitrogen in consideration of mutation by PCR. Each PCR product was found to be 369, 372, and 1494 bp, corresponding to the three gene sizes.
다. 형질전환 수행All. Perform transfection
pRSET 벡터에 BamHI, XhoI과 EcoRI 제한효소를 이용하여 PCR 산물과 벡터를 37˚C에서 2시간 이상 절단하고, 아가로즈 겔에 전기영동후 EtBr 염색을 통해 밴드 강도를 기준으로 벡터: 삽입체(insert)의 비율을 1:3으로 연결하였다. 연결(ligation) 반응후 적격 세포 (DH5α)에 형질전환시켜 클로닝된 콜로니를 확보하였다(도 6 참조). 총 22개의 콜로니를 확인한 결과 콜리니 6, 7, 8, 10, 11, 13, 18, 20, 21의 9개의 콜로니들에서 정확한 삽입이 확인되었다. 삽입이 확인된 각각의 13 kDa, 14 kDa, 및 49 kDa 재조합 단백질 발현 플라스미드를 이용하여 T7 프로모터 프라이머를 이용하여 유전자의 염기서열을 분석한 결과 100% 일치하는 것을 확인할 수 있었다.
PCR products and vectors were digested at 37 ° C for more than 2 hours using BamHI, XhoI and EcoRI restriction enzymes in the pRSET vector and electrophoresed on agarose gels, followed by EtBr staining, ) Was connected at a ratio of 1: 3. After the ligation reaction, the competent cells (DH5?) Were transformed to obtain cloned colonies (see FIG. 6). A total of 22 colonies were identified and correct insertion was confirmed in 9 colonies of
라. 13 kDa, 14 kDa, 및 49 kDa 재조합 단백질 발현의 확인la. Identification of 13 kDa, 14 kDa, and 49 kDa recombinant protein expression
13 kDa, 14 kDa, 및 49 kDa BL21 균주에 형질전환시킨 후 발현이 높은 이. 콜라이(E. coli) 콜로니를 선별하기 위해 7~8개의 콜로니를 IPTG 1mM을 이용하여 4시간 유도시킨 후 15% 비스아크릴아마이드 겔에서 90 분간 80V에서 전기영동하였다 (Loading volume: 1 ㎖ induction, 25 ㎕ 1X sample buffer Boiling 5 min, 10 ㎕ loading). 그 결과, 13-1, 14-4와 49-8번이 비교적 많이 발현되는 것으로 확인되었다(도 7 참조).
13 kDa, 14 kDa, and 49 kDa BL21 strains. To select E. coli colonies, 7 to 8 colonies were induced with 1 mM IPTG for 4 hours and then electrophoresed on a 15% bisacrylamide gel for 90 minutes at 80 V (Loading volume: 1 ml induction, 25 L 1x sample buffer Boiling 5 min, 10 l loading). As a result, it was confirmed that 13-1, 14-4 and 49-8 were relatively highly expressed (see Fig. 7).
마. 선별된 콜로니를 이용하여 다양한 조건으로의 유도(Induction) 수행hemp. Induction of various conditions using selected colonies
14 kDa 재조합 단백질의 경우 IPTG의 농도와 관계없이, 1시간 정도 IPTG 유도로 안정적 발현이 확인되었다. 3 ㎖ 콜로니를 LB 배양액에 O/N 배양 후 새로운 LB 배양액 10 ㎖에 3 ㎖ 콜로니 배양액 중 0.5 ㎖ 접종시킨 후 2시간 이상 배양 후 OD600 값이 0.5가 되면 IPTG를 이용하여 유도하고 시간대 별로 샘플을 회수하고, 그 후 15% 비스아크릴아마이드 겔에서 90 분간 80V에서 전기영동하였다 (Loading volume: 1 ㎖ induction, 25 ㎕ 1X sample buffer Boiling 5 min, 10 ㎕ loading). 13 kDa, 14 kDa, 및 49 kDa 단백질을 대장균에서 발현시키기 위해서 시간대 별로 유도를 수행한 결과 각각의 14 kDa 재조합 단백질만이 안정으로 발현됨을 확인하였다 (도 8 참조).
Regardless of the concentration of IPTG, the 14 kDa recombinant protein was stable for 1 hour with IPTG induction. 3 ml colony was O / N incubated in LB culture, 0.5 ml of 3 ml colony culture solution was inoculated in 10 ml of new LB culture medium, and incubated for 2 hours or more. After OD600 value reached 0.5, induction was carried out using IPTG. And then electrophoresed on a 15% bisacrylamide gel for 90 minutes at 80 V (Loading volume: 1 ml induction, 25 μl 1X sample buffer Boiling 5 min, loading 10 μl). In order to express 13 kDa, 14 kDa and 49 kDa proteins in E. coli, it was confirmed that only 14 kDa recombinant proteins were expressed stably (see FIG. 8).
바. 대용량 배양을 통한 p14 재조합 단백질의 분리 정제bar. Purification of p14 recombinant protein by large-scale culture
p14 단백질만 안정적으로 발현되는 결과(10 ㎖ 배양, 1mM IPTG)를 바탕으로 대용량 배양 (300 ㎖)을 실시한 결과 유도(0.1 mM IPTG)가 안정적으로 되는 것을 확인하였다 (도 9a 참조). 또한 300 ㎖ 배양된 세포를 회수하여 BUGBUSTER MASTER MIX (Merk)를 이용하여 단백질을 추출하였다. 추출된 단백질에서 HIS taq BUGBUSTER NI-NTA HIS*BIND PURIFICATION KIT (Merk)를 이용하여 2 ㎖ 중 20 ㎕를 SDS-page한 결과 정제가 되었음을 확인하였다. 정제의 순도를 확인하기 위해 정제과정중 HIS taq 컬럼에 통과되어 나온 액(Flow through), 세척과정에서 발생한 액(wash fraction)과 정제된 단백질(elution fraction)을 코마시 블루 250 염색으로 확인한 결과 정제가 양호하게 되었음을 확인하였다. 또한 동일한 방법으로 여러 차례 회수된 p14 단백질이 투석과정을 통해서 그 단백질이 잘 유지되고 있음을 확인하였다 (도 9b 참조).
As a result of stably expressing p14 protein only (10 ml culture, 1 mM IPTG), large volume culture (300 ml) was performed and it was confirmed that induction (0.1 mM IPTG) was stable (see FIG. In addition, 300 ml cultured cells were recovered and proteins were extracted using BUGBUSTER MASTER MIX (Merk). From the extracted proteins, 20 μl of 2 ml of SDS-PAGE was confirmed using HIS taq BUGBUSTER NI-NTA HIS * BIND PURIFICATION KIT (Merk). In order to confirm the purity of the tablets, the flow through, the wash fraction and the elution fraction passed through the HIS taq column during the purification process were checked with Comasy Blue 250 stain. As a result, Was found to be good. In addition, it was confirmed that the protein was maintained well through the dialysis process of the p14 protein recovered several times in the same manner (see FIG. 9B).
사. p14 재조합 단백질의 아미노산 서열분석(도 10 참조)four. Amino acid sequence analysis of p14 recombinant protein (see Fig. 10)
대장균에서 발현된 p14 재조합 단백질을 전기영동한 후에 코마시(Coomassie) 젤에서 밴드를 회수하여 Destaining 용액 (50% MeOH/D.W.) 100 ㎕를 넣고 5분간 교반한 후 Destaining 용액을 완전히 제거한다. 그리고 200 mM ABC (ammonium bicarbonate, pH7.8) 용액을 100 ㎕ 넣고 20분간 보관한 후 ABC 용액을 제거한 후, 100 ㎕의 아세토니트릴을 넣고 2분간 볼텍싱한다. 아세토니트릴 용액을 제거하고, 100 ㎕의 ABC 용액을 넣고 2분간 볼텍싱한다. ABC 용액을 제거한 후, 100 ㎕의 아세토니트릴을 넣고 2분간 볼텍싱한다. 위 과정을 총 3회 반복한다. 위 8의 마지막 단계에서 아세토니트릴을 완전히 제거하고, 상온에서 젤을 말린다. 건조된 젤에 20 ㎕의 트립신 용액 (0.2㎍)을 넣고 얼음에서 45분간 보관한다. 젤에 흡수된 트립신 용액외 나머지는 다시 제거한다. 70 ㎕의 50mM ABC 용액을 넣고, 37C 12시간 이상 보관한다. Geload Tip과 Poros R2 레진을 이용하여 마이크로 스케일의 컬럼을 제작한다. 10 ㎖의 주사기를 이용하여 마이크로 컬럼에 압력을 줄 수 있게 주사기 앞쪽을 컬럼과 일치하도록 제작한다. 제작된 컬럼에 20 ㎕의 5% 포름산 용액을 넣고 주사기를 이용하여 컬럼을 통과시킨다. (평형화) In-gel digestion에서 만들어진 펩타이드 용액 30 ㎕를 마이크로 컬럼에 넣고 주사기로 통과시킨다. (로딩) 20 ㎕의 5% 포름산 용액을 넣고 주사기를 이용하여 컬럼을 통과시킨다. 1.5 ㎕의 50% 메탄올/49% H2O/1% 포름산 용액을 넣고 주사기를 이용하여 컬럼내 펩타이드를 용출한다(세정). 1.5 ㎕의 용출물은 nanoelectrospray needle (EconoTipTM, New Objective, USA)에 넣고 Q-TOF 소스에 장착한다(elution). MS 기기: Hybrid Quadrupole-TOF LC/MS/MS Mass Spectrometer (AB Sciex Instruments, CA 94404 USA)를 이용하여 분리된 단백질의 아미노산 서열을 분석하였다. 분석결과 10, 14, 14개의 펩타이드 세개를 분석한 결과 p14 아미노산 서열과 일치함을 확인하였다(프로테인 웍스에서 수행함).
After electrophoresis of the p14 recombinant protein expressed in E. coli, the band is recovered from Coomassie gel and 100 ㎕ of the destaining solution (50% MeOH / DW) is added and stirred for 5 minutes and the destaining solution is completely removed. Then add 100 μl of 200 mM ABC (ammonium bicarbonate, pH 7.8) solution, store for 20 minutes, remove ABC solution, add 100 μl of acetonitrile and vortex for 2 minutes. The acetonitrile solution is removed and 100 μl of ABC solution is added and vortexed for 2 minutes. After removing the ABC solution, 100 μl of acetonitrile is added and vortexed for 2 minutes. Repeat the above procedure three times in total. At the end of
실험예 1. 시험관 내(In vitro) 항암 효과 확인(MTT assay)Experimental Example 1. In Vitro Antitumor Effectiveness (MTT assay)
대장암 세포주인 DLD-1과 HT-29 세포 각각을 96 웰 플레이트의 각 웰에 넣고, 본 발명의 p14 단백질을 농도별(10 ng/ml에서 1000 ng/ml)로 첨가하였다. 음성대조군으로는 0.1% PBS만을 처리하였고, 양성 대조군으로는 유산균에서 분리된 P13 단백질을 처리하였다. 이를 48시간 동안 배양한 다음, 용해 완충액(lysis buffer)으로 용해시키고, 여기에 MTT (3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide, 시그마사, 미국)가 들어있는 반응 혼합물을 넣어 37℃에서 50분간 더 배양하여 반응시켰다. 마이크로플레이트 리더(Microplate reader,Benchmark, BIO-RAD, 일본)를 사용하여 610nm에서 흡광도를 측정하여 세포 생존율(Cell survivability)을 산출하여 도 11a-11b에 나타내었다. 대장암 세포주 HT-29에 대한 결과는 도 11a에 나타내었고,대장암 세포주 DLD-1에 대한 결과는 도 11b에 나타내었다. 도 11a 및 도 11b에 도시된 바와 같이, P13 단백질은 암세포의 성장 억제 효과를 나타내지 않았으나, p14 단백질은 DLD-1 및 HT-29 세포주에 대하여 농도에 따라서 약20% 내지 약 40% 세포성장 억제율을 나타내어 탁월한 암세포 성장 억제 효과를 나타내었다. DLD-1 and HT-29 cells, which are colon cancer cell lines, were placed in each well of a 96-well plate and the p14 protein of the present invention was added at a concentration of 10 ng / ml to 1000 ng / ml. Only 0.1% PBS was treated as a negative control, and P13 protein isolated from lactic acid bacteria was treated as a positive control. This was incubated for 48 hours and then lysed with a lysis buffer. A reaction mixture containing MTT (3- (4,5-dimethylthiazol) -2,5-diphenyltetrazolium bromide (Sigma, USA) Followed by further incubation at 37 ° C for 50 minutes. The cell survivability was calculated by measuring the absorbance at 610 nm using a microplate reader (Benchmark, BIO-RAD, Japan) and is shown in FIGS. 11A and 11B. The results for the colon cancer cell line HT-29 are shown in FIG. 11A, and the results for the colon cancer cell line DLD-1 are shown in FIG. 11B. As shown in FIGS. 11A and 11B, the P13 protein did not exhibit cancer cell growth inhibitory effect, but the p14 protein inhibited cell growth inhibition rate of about 20% to about 40% depending on the concentration of DLD-1 and HT-29 cell lines And showed excellent cancer cell growth inhibitory effect.
실험예Experimental Example 2. 세포독성 테스트 2. Cytotoxicity test
본 발명의 p14 단백질의 세포독성 유무를 확인하기 위하여, MTT 분석에 의해서 세포독성 테스트를 행하였다. 대량 발현시킨 p14 단백질과 양성 대조군으로서의 P13 단백질에 대한 세포독성을 마우스 대식세포주인 Raw264.7 세포에서 확인하였다. 세포를 96 웰 플레이트의 각 웰에 5×103/well의 밀도로 넣고, 대량 발현으로 정제한 P13 및 p14 단백질을 농도별(10 ng/ml에서 1000 ng/ml)로 첨가하였다. 이를 48시간 동안 배양한 다음, 용해 완충액(lysis buffer)으로 용해시키고, 여기에 MTT (3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide, 시그마사, 미국)가 들어있는 반응 혼합물을 넣어 37℃에서 50분간 더 배양하여 반응시켰다. 마이크로플레이트 리더(Microplate reader,Benchmark, BIO-RAD, 일본)를 사용하여 610nm에서 흡광도를 측정하여 세포 생존율(Cell survivability)을 산출하여 도 12에 나타내었다. MTT 분석에 의한 세포독성 평가 결과 대조군과 비교하여 차이가 없어 p14 단백질 자체에 세포독성은 없는 것으로 확인하였다(도 12참조).
To confirm the cytotoxicity of the p14 protein of the present invention, cytotoxicity test was performed by MTT assay. Cytotoxicity against the largely expressed p14 protein and P13 protein as a positive control was confirmed in Raw264.7 cells, a mouse macrophage line. The cells were added to each well of a 96-well plate at a density of 5 × 10 3 / well and P13 and p14 proteins purified by mass expression were added by concentration (10 ng / ml to 1000 ng / ml). This was incubated for 48 hours and then lysed with a lysis buffer. A reaction mixture containing MTT (3- (4,5-dimethylthiazol) -2,5-diphenyltetrazolium bromide (Sigma, USA) Followed by further incubation at 37 ° C for 50 minutes. The cell survivability was calculated by measuring the absorbance at 610 nm using a microplate reader (Benchmark, BIO-RAD, Japan) and is shown in FIG. As a result of the cytotoxicity evaluation by MTT analysis, there was no difference compared to the control group, and it was confirmed that the p14 protein itself was not cytotoxic (see FIG. 12).
실험예 3. p14 단백질의 면역활성 확인Experimental Example 3. Confirmation of immunological activity of p14 protein
본 실험예에서는 본 발명의 p14 단백질의 항암면역 활성을 조사하기 위하여, 마우스에서 비장 세포(Balb/c splenocytes)를 적출하여 분리한 비장 세포를 2x106 세포/㎖로 96-웰 플레이트의 각 웰에 분주하였다. 표적 단백질인 P13 및 p14 단백질을 농도별(0.01㎍/㎖ 내지 10 ㎍/㎖)로 각 웰에 처리하고 48 시간 동안 배양하였다. 배양 후, 원심분리기를 이용하여 상층액만을 분리하여 ELISA를 이용하여 측정하였다. 음성대조군으로서 본 발명의 p14 단백질을 포함하지 않는 0.1% PBS 용액만을 처리하였고, 양성 대조군으로서 P13 단백질을 처리하여 비교하였다. 싸이토카인 INF-γ와 IL-12 싸이토카인의 농도(pg/㎖) 변화를 측정하여 각각 도 13a 및 도 13b에 나타내었다. 그 결과 p14 단백질은 양성 대조군인 P13 단백질에 비해 IL-12와 INF-γ의 유도능이 더 높아 항암 면역 작용에 있어 효능이 있음을 확인하였다.
In order to investigate the anti-cancer immunity activity of the p14 protein of the present invention, spleen cells (Balb / c splenocytes) were isolated from mice and splenocytes were isolated from each well of a 96-well plate at 2 × 10 6 cells / Respectively. The target proteins, P13 and p14, were treated with each concentration (0.01 쨉 g / ml to 10 쨉 g / ml) in each well and cultured for 48 hours. After culturing, only the supernatant was separated using a centrifuge and measured by ELISA. As a negative control, only the 0.1% PBS solution containing no p14 protein of the present invention was treated, and the P13 protein was treated as a positive control and compared. The changes in the concentration (pg / ml) of the cytokines INF-y and IL-12 cytokines were measured and are shown in Figs. 13A and 13B, respectively. As a result, p14 protein was more effective in the induction of IL-12 and IFN-γ compared to the positive control P13 protein.
실험예 4. 생체내(In vivo) 항암 효과 확인(MTT assay)Experimental Example 4. In vivo Antitumor Effectiveness (MTT assay)
4-1) 1,2-디메틸히드라진(DMH)에 의한 용종 유도 4-1) Polyp Induction by 1,2-dimethylhydrazine (DMH)
F344/N 쥐에 발암물질로서 1,2-디메틸히드라진(DMH; Sigma, USA)을 시트르산염 버퍼 용액에 용해하여 30 mg/kg의 용량으로 두경부에 주 2 회씩 2 주간 총 4 회 피하투여하여 용종 형성을 통하여 대장암을 유도하였다. 시험물질은 p14 단백질을 분말 형태인 기초 사료와 혼합하여 펠렛 형태로 준비하여 사용하였다.(DMH; Sigma, USA) as a carcinogen in F344 / N rats was subcutaneously administered to the head and neck at a dose of 30 mg / kg twice a week for 2 weeks for a total of 4 times, Colon cancer. The test substance was prepared in the form of a pellet by mixing the p14 protein with a powdery basic feed.
실험군은 총 4 군으로 생리식염수를 투여한 정상 쥐인 거짓대조군(Sham)과 발암물질인 DMH를 투여하고 기초사료만을 급여한 DMH 단독투여군(PC)과 발암물질 투여와 동시에 P13 단백질만 투여한 군, p14 단백질만 투여한 군 투여한 군으로 분리하였다. 음수는 정제수와 기초사료를 자유롭게 섭취하게 하였고, 시험물질은 각각 200 ug/kg의 양으로 총 8 주간 투여하였다. 실험기간 동안 주 1회 체중과 사료섭취량을 측정하여 기록하였고, 임상증상관찰을 통해 이상 유무를 확인하였다(도 14 참조).In the experimental group, DMH alone (PC) supplemented with basal diet alone, P13 protein alone group and carcinogen were administered at the same time as the normal group (Sham) and DMH (carcinogen) administered with physiological saline, p14 protein alone group. Negative water was allowed to ingest the purified water and the basic diet freely, and the test substances were administered in the amount of 200 ug / kg each for a total of 8 weeks. Body weight and feed intake were measured once a week for the duration of the experiment and recorded by observing clinical signs (Fig. 14).
도 14에 도시된 바와 같이, DMH 주입 후 대조군에 비해 모든 군에서 사료 섭취량이 현저히 감소하였다. 이러한 현상은 DMH 처리에 의한 사료 섭취 억제가, DMH에 의한 용종 발생으로 인한 체내에서의 소화 흡수 저하 때문인 것으로 추정되었다. p14 단백질을 투여한 이후부터는 대조군에 비해 점차 사료 섭취율이 증가하였으며, 특히 P13 단백질 또는 P13 단백질과 p14 단백질의 혼합물을 처리한 경우 보다도 높은 사료 섭취율을 보여 대장암의 개선 효과가 기대되었다.
As shown in FIG. 14, after DMH injection, feed intake was significantly decreased in all groups compared to the control group. This phenomenon was presumed to be due to the decrease of digestion and absorption in the body due to the polyp caused by DMH. After the administration of the p14 protein, the feed intake was gradually increased as compared with the control group. In particular, the feed intake was higher than that of the P13 protein or a mixture of the P13 protein and the p14 protein.
4-2) DMH에 의한 용종 유도 확인4-2) Confirmation of polyp induction by DMH
p14 단백질 처리에 따른 용종 억제 효과를 확인하기 위하여, 8 주간의 시험 물질 투여 후 모든 개체에 부검을 실시하였다. 항문 위 직장 부위부터 대장 조직을 일정한 크기로 절제하여 용종 형성을 확인하였다. 대장은 0.85% 식염수와 10% 중성 포르말린을 1 : 1로 혼합한 용액으로 대장을 최대한 부풀린 후, 10% 중성 포르말린 용액에 넣어 30 분간 전고정시켜서 이를 얇게 펼쳐서 모양이 뒤틀리지 않도록 여과지에 부착하여 다시 10% 중성 포르말린에 후고정하였다. 고정된 대장은 0.5% 메틸렌 블루(methylene blue)로 염색하여 개체당 발생한 선와(aberrant crypt, AC)와 선와소(aberrant crypt foci, ACF)를 고정한 대장 전체에서 ×100 광학현미경으로 육안으로 관찰하여 발생수를 기록하여 도 15a 및 도 15b에 각각 나타내었다. 도 15a 및 도 165를 통해서 확인되는 바와 같이, 선와와 선와소의 생성 수준이 대조군 보다 p14 단백질 처리군에서 유의적인 감소 효과를 나타내었다. To confirm the polyp inhibition effect of p14 protein treatment, all subjects were autopsied after 8 weeks of test substance administration. Colon tissue was resected to a certain size from the anterior rectal region to confirm polyp formation. Colon was largely infused with 0.85% saline solution and 10% neutral formalin solution in 1: 1 ratio. After the colon was maximally inflated, it was fixed in 10% neutral formalin solution for 30 minutes and spread on it thinly. And postfixed in 10% neutral formalin. The fixed colon was stained with 0.5% methylene blue and visually observed with a x100 optical microscope in the entire large intestine fixed with aberrant crypt (AC) and aberrant crypt foci (ACF) The numbers are shown in Figs. 15A and 15B, respectively. As can be seen from FIGS. 15A and 165, the level of production of line, line, and beef was significantly reduced in the p14 protein-treated group than in the control group.
실험예Experimental Example 5. 5. XenograftXenograft 모델 동물을 이용한 Using model animals p14p14 단백질의 항암활성 테스트 Anticancer activity test of protein
1 x 107 개의 사람 유래 대장암 세포주(DLD-1)를 동물 누드-마우스의 피하로 투여하여 이식시키는 Xenograft 모델을 사용하여 p14 단백질 물질의 항암 활성을 평가하였다. 이를 위해 1 x 107 세포/누드마우스/100㎕을 피하에 이식하였다. 이식 10일 후 종양세포의 이식 상태를 확인한 후 안정적인 이식 상태를 나타내는 동물에 대하여 지속적인 관찰을 실시하였다. 종양의 중심괴사(central necrosis)가 일어나기 전에 충분한 혈액공급으로 급속히 자라는 단계의 종양을 포함하는 동물을 희생시켜 주로 급속한 분열이 일어나는 외곽 부위를 일정한 크기(3×3×3 mm)로 잘라 종양절편을 제작하였다. 이어서 투관침(trocar)의 끝에 종양편(tumor fragment)을 올리고, 동물의 좌측후지 전측방을 약 4 mm 가량 절제하여 이곳을 통하여 준비한 투관침을 삽입, 좌측전지의 후방의 체간 측면부에 끝이 이르도록 하였다. 투관침을 가볍고 빠르게 360도를 회전시키며 빼주어 종양편이 목표한 위치에 자리잡도록 해주고 절제부위는 소독하였다. 피부 위를 만져 종양편의 위치를 확인하고 1주일에 2회 이상 성장을 관찰하여 성공적인 이식 상태를 나타내는 동물만을 선별하여 실험에 사용하였다. The anti-cancer activity of the p14 protein material was evaluated using a Xenograft model in which 1 x 10 7 human colorectal cancer cell lines (DLD-1) were implanted subcutaneously in animal nude-mice. For this, 1 x 10 7 cells / nude mice / 100 μl were transplanted subcutaneously. After 10 days of transplantation, the transplantation status of the tumor cells was checked, and the animals showing stable transplantation status were observed continuously. Before the central necrosis of the tumor occurs, animals that contain tumors that grow rapidly with sufficient blood supply are sacrificed and the outer area where rapid division occurs is cut to a certain size (3 × 3 × 3 mm) Respectively. Then, a tumor fragment was placed on the end of a trocar, and about 4 mm of the left side of the left side of the animal was cut off. The prepared trocar was inserted through the end of the trocar and the end was reached to the side of the rear side of the left cell . The trocar was lightly and rapidly rotated 360 degrees to allow the tumor to settle at the desired location, and the ablation site was disinfected. Only the animals that showed successful transplantation status were selected for the experiment by confirming the location of the tumor site by touching the skin and observing the growth more than once a week.
Xenograft 모델에 p14 단백질을 1 mg/kg 용량으로 2일 1회씩 총 4회 혈관투여하고, 투여 기간 중 2일 1회씩 버니어켈리퍼스를 사용하여 종양의 부피를 측정하였고, 측정된 값을 도 16에 그래프로 나타내었다. The p14 protein was administered to the Xenograft model at a dose of 1 mg / kg once a day for four times, and the volume of the tumor was measured using a vernier caliper at once every two days during the administration period. Respectively.
[수식][Equation]
종양의 부피=(장축의 길이 * (단축의 길이 * 단축의 길이))/2 Tumor volume = (length of major axis * (length of minor axis * length of minor axis)) / 2
총 4회 투여 종료 후 종양을 적출하여 사진 촬영을 실시하여 도 17에 나타내었다. After the administration of the tumor was completed four times in total, the tumor was taken and photographed, and it is shown in Fig.
도 16 및 도 17을 통해서 확인되는 바와 같이, p14 단백질 투여군에서는 완전하지는 않지만, 종양 성장 속도가 제어되어 종양 크기가 눈에 띄게 감소하였고, 대조군의 종양 부피 증가율이 63.3%인 것에 비해서, p14 단백질 투여군의 증가율은 49.6% (92.8→196.8㎣)로 10% 이상의 저해효과를 나타내었다.
As can be seen from Figs. 16 and 17, although the tumor growth rate was not completely controlled in the p14 protein administration group, the tumor size was markedly decreased and the tumor volume increase rate of the control group was 63.3% The rate of increase was 49.6% (92.8 → 196.8 ㎣), which showed an inhibitory effect of more than 10%.
본 발명은 그 사상 및 범위로부터 벗어남 없이 다양하게 변형 및 변화되어 실시될 수 있고, 이러한 사실은 당업자에게 자명할 것이다. 본 명세서에 기재된 구체적인 실시예는 단지 본 발명의 바람직한 구현예를 설명하기 위한 것으로, 본 발명을 제한하는 것으로 해석되어서는 안 된다. 본 발명의 보호범위는 첨부된 특허청구범위에 의해서 정해져야 하며, 상기의 다양한 변형 및 변화는 본 발명의 보호범위에 포함되는 것으로 의도된다. The present invention can be practiced with various modifications and changes without departing from the spirit and scope thereof, as will be apparent to those skilled in the art. The specific embodiments described herein are merely for explaining preferred embodiments of the present invention and should not be construed as limiting the present invention. The protection scope of the present invention should be defined by the appended claims, and various modifications and changes described above are intended to be included in the protection scope of the present invention.
<110> CELL BIOTECH CO., LTD. <120> Anti-Cancer Pharmaceutical Composition <130> P11-CBT-06 <160> 4 <170> KopatentIn 2.0 <210> 1 <211> 123 <212> PRT <213> Lactobacillus casei <400> 1 Met Ala Lys Ser Gln Asp Gln Phe Asn Glu Lys Val Gly Lys Glu Ile 1 5 10 15 Asn Val Ser Asp Glu Ala Val Asp Lys Ala Ala Ala Gln Ile Glu Lys 20 25 30 Val Gly Tyr Val Thr Glu Lys Asp Val Pro Glu Met Ile Asp Arg Asp 35 40 45 Tyr Thr Arg Ala Leu Ser Lys Lys Val Ser Ala Lys Leu His Lys Asp 50 55 60 Asn Asp Asp Asp Tyr Phe Tyr Glu Glu Pro Phe Asp Tyr Glu Asn Gly 65 70 75 80 Arg Ile Ala Asn Ile Met Trp Asp Met Asp Lys Ile Lys Thr Arg Glu 85 90 95 Glu Ala Met Lys Ile Leu Ala Asp Glu Leu Gly Leu Thr Val Pro Lys 100 105 110 Ile Val Met Arg Lys Ile Asp Glu Gln Val Phe 115 120 <210> 2 <211> 372 <212> DNA <213> Lactobacillus casei <400> 2 atggcaaaat ctcaagacca attcaatgaa aaggtcggta aagaaattaa cgtttctgac 60 gaggccgtcg ataaagcagc tgcccaaatc gagaaagtcg gttacgtcac tgaaaaggac 120 gttccggaaa tgatcgatcg cgattacact cgggcattgt ccaagaaggt ttcggcaaaa 180 cttcacaaag acaatgacga tgattacttc tacgaagagc cttttgacta tgaaaatggc 240 cgcattgcaa atatcatgtg ggacatggat aaaattaaga cccgcgaaga agccatgaaa 300 attttggctg atgaacttgg ccttaccgtt cctaagattg taatgcgtaa aatcgacgaa 360 caggtcttct ag 372 <210> 3 <211> 123 <212> PRT <213> Lactobacillus rhamnosus GG <400> 3 Met Ala Lys Ser Gln Asp Gln Phe Asn Glu Lys Ala Gly Lys Lys Ile 1 5 10 15 Thr Val Ser Asp Glu Ala Val Asp Lys Ala Ala Lys Lys Ile Glu Gln 20 25 30 Val Gly Tyr Val Thr Glu Lys Asp Val Pro Glu Met Ile Asp Arg Asp 35 40 45 Tyr Thr Arg Ala Leu Ser Lys Lys Val Ser Ala Lys Leu His Gln Asp 50 55 60 Lys Asp Asp Asp Tyr Phe Tyr Glu Glu Pro Phe Asp Tyr Glu Asn Gly 65 70 75 80 Arg Ile Ala Asn Ile Ile Trp Asp Met Asp Lys Ile Lys Thr Arg Glu 85 90 95 Glu Ala Met Lys Thr Leu Ala Asn Glu Leu Gly Leu Thr Val Pro Lys 100 105 110 Ile Val Met Arg Lys Val Asp Glu Gln Val Phe 115 120 <210> 4 <211> 372 <212> DNA <213> Lactobacillus rhamnosus <400> 4 atggcaaaat ctcaagacca attcaatgaa aaggcgggca agaagattac agtttccgat 60 gaggctgtcg ataaggccgc caaaaaaatt gaacaggtcg gctacgtaac cgaaaaagac 120 gttccggaaa tgatcgatcg cgattatacg cgggcattgt caaaaaaggt ttccgctaag 180 ctgcaccaag acaaagacga tgactatttc tacgaggaac cttttgatta tgaaaatggc 240 cgcattgcca acatcatctg ggatatggat aaaatcaaaa cccgtgaaga agcaatgaag 300 accttagcaa atgaacttgg cttgactgtt ccaaagattg tcatgcgcaa agtcgatgaa 360 caggttttct aa 372 <110> CELL BIOTECH CO., LTD. <120> Anti-Cancer Pharmaceutical Composition <130> P11-CBT-06 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 123 <212> PRT <213> Lactobacillus casei <400> 1 Met Ala Lys Ser Gln Asp Gln Phe Asn Glu Lys Val Gly Lys Glu Ile 1 5 10 15 Asn Val Ser Asp Glu Ala Val Asp Lys Ala Ala Ala Gln Ile Glu Lys 20 25 30 Val Gly Tyr Val Thr Glu Lys Asp Val Pro Glu Met Ile Asp Arg Asp 35 40 45 Tyr Thr Arg Ala Leu Ser Lys Lys Val Ser Ala Lys Leu His Lys Asp 50 55 60 Asn Asp Asp Asp Tyr Phe Tyr Glu Glu Pro Phe Asp Tyr Glu Asn Gly 65 70 75 80 Arg Ile Ala Asn Ile Met Trp Asp Met Asp Lys Ile Lys Thr Arg Glu 85 90 95 Glu Ala Met Lys Ile Leu Ala Asp Glu Leu Gly Leu Thr Val Pro Lys 100 105 110 Ile Val Met Arg Lys Ile Asp Glu Gln Val Phe 115 120 <210> 2 <211> 372 <212> DNA <213> Lactobacillus casei <400> 2 atggcaaaat ctcaagacca attcaatgaa aaggtcggta aagaaattaa cgtttctgac 60 gaggccgtcg ataaagcagc tgcccaaatc gagaaagtcg gttacgtcac tgaaaaggac 120 gttccggaaa tgatcgatcg cgattacact cgggcattgt ccaagaaggt ttcggcaaaa 180 cttcacaaag acaatgacga tgattacttc tacgaagagc cttttgacta tgaaaatggc 240 cgcattgcaa atatcatgtg ggacatggat aaaattaaga cccgcgaaga agccatgaaa 300 attttggctg atgaacttgg ccttaccgtt cctaagattg taatgcgtaa aatcgacgaa 360 caggtcttct ag 372 <210> 3 <211> 123 <212> PRT <213> Lactobacillus rhamnosus GG <400> 3 Met Ala Lys Ser Gln Asp Gln Phe Asn Glu Lys Ala Gly Lys Lys Ile 1 5 10 15 Thr Val Ser Asp Glu Ala Val Asp Lys Ala Ala Lys Lys Ile Glu Gln 20 25 30 Val Gly Tyr Val Thr Glu Lys Asp Val Pro Glu Met Ile Asp Arg Asp 35 40 45 Tyr Thr Arg Ala Leu Ser Lys Lys Val Ser Ala Lys Leu His Gln Asp 50 55 60 Lys Asp Asp Tyr Phe Tyr Glu Glu Pro Phe Asp Tyr Glu Asn Gly 65 70 75 80 Arg Ile Ala Asn Ile Ile Trp Asp Met Asp Lys Ile Lys Thr Arg Glu 85 90 95 Glu Ala Met Lys Thr Leu Ala Asn Glu Leu Gly Leu Thr Val Pro Lys 100 105 110 Ile Val Met Arg Lys Val Asp Glu Gln Val Phe 115 120 <210> 4 <211> 372 <212> DNA <213> Lactobacillus rhamnosus <400> 4 atggcaaaat ctcaagacca attcaatgaa aaggcgggca agaagattac agtttccgat 60 gaggctgtcg ataaggccgc caaaaaaatt gaacaggtcg gctacgtaac cgaaaaagac 120 gttccggaaa tgatcgatcg cgattatacg cgggcattgt caaaaaaggt ttccgctaag 180 ctgcaccaag acaaagacga tgactatttc tacgaggaac cttttgatta tgaaaatggc 240 cgcattgcca acatcatctg ggatatggat aaaatcaaaa cccgtgaaga agcaatgaag 300 accttagcaa atgaacttgg cttgactgtt ccaaagattg tcatgcgcaa agtcgatgaa 360 caggttttct aa 372
Claims (9)
An anticancer pharmaceutical composition comprising p14 protein comprising an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3 as an active ingredient.
According to claim 1, wherein the protein is Lactobacillus Casei ( Lactobacillus casei ), Lactobacillus paracasei ) or Lactobacillus rhamnosus ) anti-cancer pharmaceutical composition, characterized in that the protein.
The anticancer pharmaceutical composition of claim 1, wherein the protein has a molecular weight of about 14 kDa.
4. The anticancer pharmaceutical composition according to any one of claims 1 to 3, wherein the composition further comprises a pharmaceutically acceptable excipient, diluent, carrier or buffer in addition to the p14 protein.
According to claim 1, wherein the cancer is colon cancer, lung cancer, small cell lung cancer, stomach cancer, liver cancer, hematologic cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal muscle Cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, Cancers such as prostate cancer, chronic or acute leukemia, lymphocyte lymphoma, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brain stem glioma, pituitary adenoma Anticancer pharmaceutical composition, characterized in that one or more combinations of these.
An anticancer pharmaceutical composition comprising the nucleic acid encoding the p14 protein of claim 1 as an active ingredient.
The anticancer pharmaceutical composition according to claim 6, wherein the nucleic acid comprises a nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4.
7. The anticancer pharmaceutical composition according to claim 6, wherein the nucleic acid is contained in an expression vector.
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| EP12851727.3A EP2751129B1 (en) | 2011-11-23 | 2012-08-29 | Protein p14 with anti-cancer and anti-allergy activity and pharmaceutical composition comprising the same |
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| WO2022265447A1 (en) * | 2021-06-18 | 2022-12-22 | 한국생명공학연구원 | Composition comprising lacticaseibacillus sp. or lactobacillus sp. strain as active ingredient for diagnosis, prevention, or treatment of cancer |
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| WO2022265447A1 (en) * | 2021-06-18 | 2022-12-22 | 한국생명공학연구원 | Composition comprising lacticaseibacillus sp. or lactobacillus sp. strain as active ingredient for diagnosis, prevention, or treatment of cancer |
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