KR100286366B1 - Lectin genes from Viscum album - Google Patents
Lectin genes from Viscum album Download PDFInfo
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- KR100286366B1 KR100286366B1 KR1019990041288A KR19990041288A KR100286366B1 KR 100286366 B1 KR100286366 B1 KR 100286366B1 KR 1019990041288 A KR1019990041288 A KR 1019990041288A KR 19990041288 A KR19990041288 A KR 19990041288A KR 100286366 B1 KR100286366 B1 KR 100286366B1
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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Abstract
본 발명은 한국산 겨우살이 식물로부터 분리된 면역증강 기능을 갖는 렉틴 단백질 및 그 암호화 유전자에 관한 것으로, 본 발명 한국산 겨우살이 식물로부터 분리한 두가지 렉틴 단백질은 유럽산 겨우살이로부터 분리한 렉틴 단백질들과는 아미노산 서열 및 그 암호화 유전자의 염기서열에 차이가 존재하였으며, 항원-특이적 면역반응을 증강시키는 매우 뛰어난 효과가 있어 본 발명은 생물의약산업상 매우 유용한 발명인 것이다.The present invention relates to a lectin protein having an immuno-enhancing function isolated from Korean mistletoe plants and its coding genes. The two lectin proteins isolated from the Korean mistletoe plants have an amino acid sequence and its coding gene from lectin proteins isolated from European mistletoe. There was a difference in the nucleotide sequence of the present invention, which has a very excellent effect of enhancing the antigen-specific immune response is a very useful invention in the biopharmaceutical industry.
Description
본 발명은 한국산 겨우살이 식물로부터 분리된 면역증강 기능을 갖는 렉틴 단백질 및 그 암호화 유전자에 관한 것이다.The present invention relates to a lectin protein having an immune enhancing function isolated from a Korean mistletoe plant and its coding gene.
겨우살이(mistletoe. Viscum album)는 여러 종류의 나무를 숙주로 하여 생장하는 반기생식물로서 세계 전역에는 30속 1,500종의 식물이 있는 것으로 알려져 있다. 여러종류의 겨우살이 중에 현재 약재로 사용되고 있는 겨우살이는 Viscum 속(genus)의 겨우살이로서, 주로 유럽지역에서 서식되는 유럽산 겨우살이(Viscum album Loranthacea)이다. 유럽의 loranthacea과에 속하는 겨우살이는 오래전부터 민간요법으로서 고혈압, 동맥경화증, 암 등에 대하여 효과가 있는 신비의 약제로 사용되다가, 1921년부터 항암활성을 인정받아 종양에 대한 치료 및 그 보조제로 사용되고 있다. 따라서, 겨우살이의 생물학적 활성에 대한 연구는 주로 유럽에서 유럽지역에 서식하는 겨우살이에 대하여 수행되어 왔다.Mistletoe (Misttletoe.Viscum album) is a semi-parasitic plant that grows as a host of various kinds of trees and is known to have 30 plants and 1,500 species. Mistletoe, which is currently used as a medicinal herb among various kinds of mistletoe, is a mistletoe of the genus Viscum, a European mistletoe (Viscum album Loranthacea) mainly in Europe. Mistletoe, which belongs to the European loranthacea family, has been used as a folk medicine for a long time as a mysterious drug that is effective against hypertension, arteriosclerosis, and cancer. Since 1921, it has been recognized as an anticancer activity and used as an adjunct to the treatment of tumors. Therefore, studies on the biological activity of mistletoe have been carried out mainly on the mistletoe inhabiting Europe.
유럽지역에 서식하는 겨우살이는 체액성 및 세포성 면역체계를 자극하는 면역증강 효과가 있는 것으로 보고되었고, 또한 동물 및 인간에 대한 임상 실험결과 종양 세포에 대하여 직접, 간접적으로 대응하는 대식세포 및 자연살해세포의 활성을 증가시킴으로서 종양세포의 성장을 억제하여 암환자의 생존율을 높이는 효과가 있음이 보고되었다(USP 5547674). 이러한 효과는 겨우살이의 면역증강 작용 및 종양세포에 대한 직접적인 세포독성 효과에 의한 것으로 알려져 있으며, 대표적인 활성물질로는 렉틴 성분들로 당쇄부분의 특이성 및 분자량에 따라 렉틴Ⅰ, Ⅱ, Ⅲ의 3종류로 구별하고 있으며 주로 렉틴Ⅰ에 대한 면역학적, 생화학적 연구가 진행되고 있다.Mistletoe living in Europe has been reported to have an immune enhancing effect on the humoral and cellular immune systems, and clinical trials in animals and humans have shown that macrophages and natural killing of tumor cells directly and indirectly. Increasing cell activity has been reported to inhibit the growth of tumor cells to increase the survival rate of cancer patients (USP 5547674). These effects are known to be due to the immune enhancing action of mistletoe and direct cytotoxic effects on tumor cells. Representative active substances are lectins, which are classified into three types of lectins I, II, and III according to the specificity and molecular weight of sugar chains. Differentiation, mainly immunological and biochemical studies of lectin I are in progress.
한편, 유럽산 겨우살이와 구별되는 한국산 겨우살이 역시 민간 및 한방에서 요통, 고혈압, 유산방지, 치통에 대한 약재로 사용되어 왔으며, 한방에서 숙주나무에 따라 상기생, 기생목, 해기생 등의 각기 다른 이름으로 불리어진 것으로 보아 숙주나무에 따른 상이한 약효 및 그에 기인되는 성분상의 차이가 있음이 암시되었다. 실제로 유럽산 겨우살이는 숙주에 따른 렉틴성분등에서 차이가 보고된 바 있으므로 한국산 겨우살이에서 숙주등에 따른 성분의 변화가 있을 수 있다는 가능성을 배제할 수 없다. 이러한 사실에 기초하여 한국산 겨우살이(V. album, Coloratum)에 대한 과학적인 연구로서는 최초로 Khwaja 등은 한국산 겨우살이의 항종양효과 및 활성성분에 대하여 보고하였다(USP No. 5,565,200). 이 연구는 한국산 겨우살이의 항종양 효과는 유럽산의 경우와는 다르게 렉틴에 의한 것이 아니라, 강한 독성의 alkaloids에 의한 세포독성효과에 의한 것으로 보고하여 유럽산 겨우살이와 한국산 겨우살이의 항종양활성 성분 및 기작을 구별하였으나 후속 연구는 아직 없는 실정이다. 그러나, 최근의 연구결과 한국산 겨우살이 역시 macrophage를 직접 활성화 시켜서 Interleukin-1(IL-1)과 Turmor necrosis factor-α(TNF-α)를 유도하며, 이들 cytokine들을 유도하는 활성성분은 ammonium sulphate에 의해 침전되는 단백질 성분인 것으로 밝혀져 한국산 겨우살이 성분중에서도 렉틴이 존재함을 예측할 수 있었고 그후 한국산 겨우살이에서도 렉틴이 분리되었다.Meanwhile, Korean mistletoe, which is distinguished from European mistletoe, has also been used as a medicine for low back pain, hypertension, miscarriage, and toothache in private and oriental medicines. It was suggested that there are different effects on the host tree and the difference in the components resulting therefrom. In fact, European mistletoe has been reported in lectin components according to the host, so the possibility that there may be a change in the component according to the host in Korea mistletoe can not be excluded. Based on this fact, Khwaja et al. Reported the antitumor effect and active ingredient of Korean mistletoe for the first time in the scientific study on Korean mistletoe (V. album, Coloratum) (USP No. 5,565,200). This study reports that the anti-tumor effect of Korean mistletoe is not caused by lectin, but rather by cytotoxic effect by strong toxic alkaloids, unlike the case of European mistletoe, and distinguishes the antitumor activity and mechanisms of European mistletoe and Korean mistletoe. However, there is no follow-up study yet. However, recent studies have shown that Korean mistletoe also directly activates macrophage, inducing Interleukin-1 (IL-1) and Turmor necrosis factor-α (TNF-α), and the active ingredients that induce these cytokines are precipitated by ammonium sulphate. It was found to be a protein component, and it was predicted that lectin was present among Korean mistletoe components, and then lectins were isolated from Korean mistletoe.
본 발명자들은 한국산 겨우살이 식물로부터 크로마토그래피법 및 단일클론항체 선별법을 이용하여 특성이 다른 두가지의 렉틴 성분(KML-ⅡU 및 KML-ⅡL)을 분리한 후 유전자 클로닝에 의하여 렉틴 단백질 암호화 유전자의 염기서열 및 그로부터 추론된 아미노산 서열을 결정하고, 상기 분리된 두 렉틴 단백질과 유럽산 렉틴 단백질을 비교한 결과, 한국산과 유럽산의 명백한 차이점을 확인하고 렉틴 단백질의 면역증강 효과를 확인함으로써 본 발명을 완성하게 되었다.따라서, 본 발명의 목적은 한국산 겨우살이로부터 분리 정제된 면역증강 기능을 갖는 렉틴 단백질 및 그 암호화 유전자를 제공하는 것이다.We isolated two lectin components (KML-IIU and KML-IIL) with different characteristics from Korean mistletoe plants using chromatography and monoclonal antibody screening, and then cloned the nucleotide sequence of the lectin protein coding gene by gene cloning. Determining the amino acid sequence deduced therefrom and comparing the two isolated lectin proteins with European lectin proteins, the present invention was completed by confirming the apparent difference between Korean and European and confirming the immunopotentiating effect of the lectin protein. It is an object of the present invention to provide a lectin protein and its coding gene having immuno-enhancing function which is purified from Korean mistletoe.
이하, 본 발명의 구체적인 구성을 상세히 설명한다.Hereinafter, the specific configuration of the present invention will be described in detail.
도 1은 환원, 비환원 조건하에서 KML-C의 SDS-PAGE 분석을 나타낸 사진이다.1 is a photograph showing the SDS-PAGE analysis of KML-C under reducing and non-reducing conditions.
도 2는 단일클론(9H7D10)을 사용한 KML-C의 Western blot 분석을 나타낸 사진이다.Figure 2 is a photograph showing the Western blot analysis of KML-C using a monoclonal (9H7D10).
도 3은 환원 조건하에서 KML-C, KML-ⅡU, KML-ⅡL를 SDS-PAGE 분석한 사진이다.Figure 3 is a SDS-PAGE analysis of KML-C, KML-IIU, KML-IIL under reducing conditions.
도 4는 한국산 겨우살이 렉틴의 N-terminal 아미노산 서열을 나타낸 것이다.Figure 4 shows the N-terminal amino acid sequence of Korean mistletoe lectin.
도 5는 재조합 플라스미드 벡터의 모식도를 나타낸 것이다.5 shows a schematic of the recombinant plasmid vector.
도 6은 본 발명 한국산 겨우살이 렉틴 KML-IIUA 및 KML-ULB와 유럽산 겨우살이 렉틴의 일부 아미노산 서열을 비교한 것이다.Figure 6 compares the amino acid sequence of some of the present invention Korean mistletoe lectin KML-IIUA and KML-ULB and European mistletoe lectin.
도 7은 KML-ⅡU를 주사한 쥐의 비장세포의 mitogen 첨가에 대한 감응을 나타낸 그림이다.Figure 7 shows the response to mitogen addition of splenocytes of mice injected with KML-IIU.
도 8은 대식세포의 KML-ⅡU에 의한 IL-1 유도활성을 나타낸 그림이다.8 is a diagram showing the IL-1 induced activity of KML-IIU of macrophages.
도 9는 NK-세포활성 유도에 대한 KML-ⅡU의 효과를 나타낸 그림이다.9 is a diagram showing the effect of KML-IIU on the induction of NK-cell activity.
도 10은 항원 KLH에 특이적인 항체의 유도능에 대한 KML-ⅡU의 효과를 나타낸 그림이다.10 is a diagram showing the effect of KML-IIU on the inducibility of antibodies specific for antigen KLH.
도 11은 항원 KLH 단독 또는 KML-ⅡU와 동시에 면역시킴으로서 유도되는 KLH에 대한 항체의 subisotypes 분석을 나타낸 그림이다.Figure 11 shows the analysis of subisotypes of antibodies against KLH induced by immunization with antigen KLH alone or simultaneously with KML-IIU.
도 12는 항원 KLH로 면역된 마우스의 비장세포의 항원 특이적 분화를 나타낸 그림이다.12 shows antigen specific differentiation of splenocytes of mice immunized with antigen KLH.
도 13은 KLH 항원에 감작된 마우스의 비장세포로부터 KLH에 대한 IL-2와 IL-4의 생산을 나타낸 그림이다.Figure 13 shows the production of IL-2 and IL-4 against KLH from splenocytes of mice sensitized by KLH antigen.
도 14는 DTH 유도에 대한 KML-ⅡU의 효과를 나타낸 그림이다.14 shows the effect of KML-IIU on DTH induction.
본 발명자들은 한국산 겨우살이로부터 두 가지 렉틴 단백질을 분리 추출하고 출하고 클로닝한 결과 종래 식물의 렉틴 단백질들과 마찬가지로 한국산 겨우살이의 경우에도 클론간에 염기서열의 다소간의 차이를 보여 여러 isoform이 존재할 가능성이 있음을 알 수 있었다. 또한, DNA 염기서열로부터 아미노산 서열을 추론하여 기존에 알려진 유럽산 겨우살이 렉틴 단백질 및 연관된 다른 식물의 렉틴 단백질의 아모노산 서열과 비교한 결과는 서열상 차이점이 존재함을 알 수 있었다.The present inventors have isolated, extracted and cloned two lectin proteins from Korean mistletoe. As with the lectin proteins of conventional plants, we found that there is a possibility that several isoforms may exist due to the slight differences in nucleotide sequences between clones even in Korean mistletoe. Could know. In addition, the amino acid sequence was deduced from the DNA sequence and compared with the known amino acid sequence of the European mistletoe lectin protein and the related plant lectin protein.
분리한 한국산 겨우살이 렉틴의 특성조사를 위해 유럽산 겨우살이 렉틴(EML-1)을 대조군으로 하여 다양한 세포주에 대한 세포독성 효과를 시험관내에서 조사한 결과, 렉틴 단백질 KML-ⅡU는 6-BL6 멜라노마, Meth A Fibrosarcoma 세포주에서 비교적 높은 저항성을 나타낸 반면, 3LL 카시노마, Raji 림포마에 대해서는 비교적 높은 감수성을 나타내어 종양세포에 대한 일부 특이성이 있는 것으로 생각되었으며, 대부분의 종양세포에 있어서 KML-ⅡU의 세포독성 효과는 유럽산 렉틴보다 강한 것으로 나타났고, 세포주에 따라서는 10배 이상 높은 세포독성 효과를 나타내어 한국산 겨우살이로부터 분리한 렉틴 단백질의 활성이 매우 우수함을 알 수 있었다. colon26-M3.1 카시노마, B16-BL6 멜라노마 및 L1210 루케미아 세포주에서 KML-ⅡU 및 KML-ⅡL의 세포독성 효과를 비교한 결과 모든 경우에서 KML-ⅡU가 비교적 높은 세포독성 효과를 나타내었다.To investigate the characteristics of isolated Korean mistletoe lectin, the cytotoxic effect on various cell lines was investigated in vitro using European mistletoe lectin (EML-1) as a control. The lectin protein KML-IIU was 6-BL6 melanoma, Meth A. While it showed relatively high resistance in Fibrosarcoma cell line, it showed relatively high sensitivity to 3LL casinoma and Raji lymphoma, and it was thought to have some specificity for tumor cells. It was found to be stronger than European lectin, and the cell line showed more than 10 times higher cytotoxic effect, indicating that the activity of lectin protein isolated from Korean mistletoe was very good. Comparing the cytotoxic effects of KML-IIU and KML-IIL in colon26-M3.1 casinoma, B16-BL6 melanoma, and L1210 Lucemia cells, KML-IIU showed a relatively high cytotoxic effect in all cases.
이하 본 발명의 구체적인 구성 및 작용효과를 실시예를 통하여 설명하고자 한다. 단, 하기 실시예는 본 발명을 예시하기 위한 목적일 뿐 본 발명의 권리범위는 하기 실시예에 한정되는 것은 아니다.Hereinafter will be described the specific configuration and effect of the present invention through the examples. However, the following examples are only for the purpose of illustrating the present invention and the scope of the present invention is not limited to the following examples.
실시예 1. 겨우살이의 준비 및 Lectin 분리Example 1 Preparation of Mistletoe and Lectin Separation
Lectin 성분의 분리를 위해 참나무를 숙주로 하여 자라는 겨우살이를 12월~2월중에 채취하여 증류수로 3회 세척한 후 -80℃에서 동결하고 잘게 세절한 후 블렌더(blender)에서 다시 chopping한 후 5배 부피의 0.15M NaCl 용액으로 8~12시간동안 4℃에서 추출하고 10,000×g에서 20분간 원심분리 시킨후 상등액을 취하였다. 그리고 단백성분을 침전시키기 위하여 상등액에 ammonium sulfate powder를 첨가시켜 70%의 황산 암모늄 포화용액을 만들고 4℃에서 약하게 교반시켰다. 다시 10,000×g에서 20분간 원심분리시킨후 상등액은 버리고 팰렛을 취하여 최소 부피의 0.15M NaCl이 함유된 phosphate 완충용액(PBS)에 재현탁시키고 동일한 용액에서 24시간 동안 2, 3회 완충용액을 바꿔가면서 투석시켰다. 그후 10,000×g에서 원심분리 시킨후 상등액만을 취하여 0.45um membrane filter에서 여과한 후 여액을 50℃에서 3시간동안 0.2M HCl로 가수분해시킨 Sepharose 4B를 충진한 칼럼으로 통과시키고 5배 column volumn의 PBS로 세척하고 칼럼에 결합된 물질은 50mM Galactose가 함유된 PBS로 용출시켜 얻어진 부분을 KML-C로 명명하였다.Mistletoe grown with oak as a host to separate the lectin components was collected in December-February, washed three times with distilled water, frozen at -80 ℃, finely chopped, and chopping again in a blender. Extracted at 8 ° C. for 8-12 hours with a volume of 0.15 M NaCl solution, centrifuged at 10,000 × g for 20 minutes, and the supernatant was taken. In addition, ammonium sulfate powder was added to the supernatant to precipitate the protein component, and a saturated ammonium sulfate solution of 70% was made and stirred gently at 4 ° C. Centrifuge again at 10,000 × g for 20 minutes, discard the supernatant, take the pallet, resuspend in phosphate buffer (PBS) containing 0.15 M NaCl in the minimum volume, and change buffer two or three times in the same solution for 24 hours. Dialysis as it went. After centrifugation at 10,000 × g, only the supernatant was collected, filtered through a 0.45um membrane filter, and the filtrate was passed through a column filled with Sepharose 4B, hydrolyzed with 0.2M HCl at 50 ° C. for 3 hours, followed by PBS of 5-fold column volumn. The material obtained by washing with the column and eluting with PBS containing 50 mM Galactose was named KML-C.
실시예 2. KML-C의 순도 및 분자량 조사Example 2. Investigation of Purity and Molecular Weight of KML-C
KML-C의 분자량 및 순도를 측정하기 위하여 0.1% SDS를 함유하는 13% 폴리아크릴아미드 겔에서 전기영동을 실시하여 표준단백들과 비교하였다. 전개완료 후 단백 band들은 coomassie blue 염색시약으로 염색하였다. 실험결과 사진도 1에서 확인되는 바와 같이 2-mercaptoethanol을 처리한 환원상태에서는 각각 33.2, 31, 28.6, 25.5 KD의 분자량을 가지는 4개의 band가 확인되었다. 따라서 KML-C에는 두 개의 subunit을 가진 두 개의 단백질이 존재하고 있음을 확인하였으며 각각의 단백질을 KML-ⅡU(61.8KD)와 KML-ⅡL(56.4KD)로 명명하였다.To determine the molecular weight and purity of KML-C, electrophoresis was performed on 13% polyacrylamide gels containing 0.1% SDS to compare with standard proteins. After completion of development, protein bands were stained with coomassie blue dye. As a result of the experiment, as shown in FIG. 1, four bands having a molecular weight of 33.2, 31, 28.6, and 25.5 KD were observed in the reduced state treated with 2-mercaptoethanol. Therefore, it was confirmed that two proteins with two subunits exist in KML-C. Each protein was named as KML-IIU (61.8KD) and KML-IIL (56.4KD).
실시예 3. KML-C에 대한 단일클론 항체의 생산 및 특성 조사Example 3. Production and Characterization of Monoclonal Antibodies Against KML-C
단일클론 항체의 생산을 위하여 Balb/c mouse에 PBS에 20μg/100μl의 수준으로 용해시킨 KML-C를 동량의 complete Freund's adjuvant에 유화시켜 복강에 면역하였다. 14일후에 다시 1회 추가 면역한 후 10일 후에 mouse의 눈으로부터 소량의 혈액을 채취하여 혈청을 얻고 항원을 고정시킨 indirect ELISA로 역가를 확인한 후 adjuvant없이 20μg의 항원을 복강에 두 번째 주사하였다. 최종 면역 1주일 후에 Balb/c 마우스의 비장세포와 P4U1 골수종을 PEG를 이용하여 융합후 HAT 배지로 hybridoma를 선별하고 KML-C에 대한 항체를 생산하는 세포를 ELISA를 통해 선택하여 96 well plate에서 클로닝한 후 다시 screening과 클로닝을 반복하여 KML-C에 대한 단일클론 항체를 생산하는 hybridoma를 선별하였다. 항체를 대량생산하기 위하여 선택된 hybridoma들을 pristane을 주입한 후 마우스의 복강으로 주사하고 ascitic fluid를 얻었으며 항체를 정제하기 위하여 이 ascitic fluid를 protein-G column에 통과시켜 항체를 분리하였다. 정제된 단일클론 항체들 중에서 KML-C와 특이적으로 반응하는 항체를 선별하기 위하여 western blotting을 실시하였다. 먼저 KML-C를 SDS존재하에서 전기영동을 실시하고 Nitrocellulose membrane로 단백부분을 transfer시키고 각각의 단일클론항체를 반응시킨후 alkaline phosphatase가 conjugate된 2차 항체를 반응시킨후 BCIP-NBT 용액으로 발색하였다. 실험결과 사진 도 2와 같이 대조군으로 사용한 유럽산 lectin에는 결합하지 않으면서 KML-ⅡU의 윗부분(33KD)의 band에 강한 specificity를 가진 단일클론(9H7D10)을 선별하였다.For production of monoclonal antibodies, KML-C dissolved in PBS in Balb / c mice at a level of 20 μg / 100 μl was emulsified in the same amount of complete Freund's adjuvant to immunize the abdominal cavity. After another immunization 14 days later, 10 days later, a small amount of blood was collected from mouse eyes, serum was obtained, and titer was confirmed by indirect ELISA immobilized with antigen. Then, 20 μg of antigen was injected into the abdominal cavity without adjuvant. One week after the final immunization, splenocytes of Balb / c mice and P4U1 myeloma were fused using PEG, hybridomas were selected with HAT medium, and cells producing antibodies against KML-C were cloned in 96 well plates. Then, screening and cloning were repeated to select hybridomas producing monoclonal antibodies against KML-C. In order to mass-produce the antibody, selected hybridomas were injected with pristane and injected into the abdominal cavity of mice to obtain ascitic fluid. The antibody was isolated by passing the ascitic fluid through a protein-G column to purify the antibody. Western blotting was performed to select antibodies that specifically react with KML-C from the purified monoclonal antibodies. First, KML-C was subjected to electrophoresis in the presence of SDS, transfer of the protein portion to the Nitrocellulose membrane, reaction of each monoclonal antibody, and reaction of the secondary antibody conjugated with alkaline phosphatase, followed by color development with BCIP-NBT solution. As a result of the experiment, as shown in FIG. 2, a single clone (9H7D10) having strong specificity was selected for the band of the upper portion (33KD) of KML-IIU without binding to the European lectin used as a control.
실시예 4. Immuno-affinity column의 준비 및 KML-ⅡU, KML-ⅡL의 분리Example 4 Preparation of Immuno-affinity Columns and Isolation of KML-IIU and KML-IIL
KML-ⅡU와 KML-ⅡL 두 개의 단백질을 분리하기 위하여 9H7D10 항체를 제조사(Pharmacia Biotech)의 지침에 따라 activated Sepharose로 고정시킨 immuno-affinity column을 만들었으며 이를 이용하여 다음과 같은 방법에 따라 두 개의 단백질을 분리하였다. 먼저 KML-C를 PBS에서 투석하여 갈락토오스를 제거한 후 immuno affinity column으로 통과시키고 PBS로 용출되는 부분(KML-ⅡL)과 glycine-HCl buffer(pH 2.7)에 용출되는 부분을(KML-ⅡU) 각각 수집하고 전기영동을 통해 순도를 측정하고 대조군으로서 유럽산 겨우살이 lectin(EML)과 비교하였다. 실험결과는 사진도 3과 같이 KML-C로부터 KML-ⅡU와 KML-ⅡL를 각기 분리할 수 있었으며 또한 이들이 EML과는 각기 다른 분자량을 가진 단백 band들로 확인되었다.In order to separate the two proteins KML-IIU and KML-IIL, an immuno-affinity column was immobilized with 9H7D10 antibody activated Sepharose according to the manufacturer's (Pharmacia Biotech's) instructions. Was separated. First, KML-C was dialyzed in PBS to remove galactose, then passed through an immuno affinity column, and the parts eluted with PBS (KML-IIL) and the parts eluted with glycine-HCl buffer (pH 2.7) (KML-IIU) were collected, respectively. Purity was measured by electrophoresis and compared with European mistletoe lectin (EML) as a control. Experimental results showed that KML-IIU and KML-IIL could be separated from KML-C as shown in Figure 3, and they were identified as protein bands with different molecular weight from EML.
실시예 5. KML-ⅡU와 KML-ⅡL에 대한 적혈구 응집반응실험 및 당저해반응실험Example 5 Hemagglutination and Glycolysis Reaction Test against KML-IIU and KML-IIL
분리된 한국산 겨우살이 lectin들이 agalutinin으로서의 기능을 가지는지, 그리고 어떠한 당에 specificity를 가지는지에 대해 조사하고자 적혈구 응집반응과 당저해반응을 실시하였다. 응집반응과 저해반응 test는 U-Type 96 well microtiter plate를 이용하여 연속적인 두배 희석법으로 실시하였으며 그 과정은 다음과 같다. 먼저 B Type의 적혈구를 취하여 PBS에서 2%가 되게 현탁시킨후 정해진 농도의 lectin solution과 96 well microtiter plate에서 1시간 동안 배양하여 응집을 위해 요구되는 최소의 농도를 확인하였다(표 1). 그리고 이 농도의 lectin solution을 다양한 종류 및 다양한 농도의 당과 30분간 배양한 후 다시 적혈구 현탁액과 반응시켜 당에 의한 응집저해여부를 관찰하였다(표 2). 실험결과, KML-C와 KML-ⅡU 및 KML-ⅡL 공히 8μg/mL 수준에서 응집반응이 관찰되었고 Lactose, galactose, N-acetylgalactoseamine에 의해서 응집효과가 저해되었다. 따라서, 본 발명에서 분리된 한국산 lectin들은 Lactose, galactose, N-acetylgalactoseamine에 specipicity를 가지는 lectin으로 확인되었다.In order to investigate whether isolated Korean mistletoe lectins function as agalutinin and which sugars have specificity, erythrocyte aggregation and glucose-inhibition reactions were performed. Agglutination and inhibition reactions were performed by serial double dilution using a U-Type 96 well microtiter plate. First, B type red blood cells were suspended in 2% PBS, and then cultured for 1 hour in a predetermined concentration of lectin solution and 96 well microtiter plate to confirm the minimum concentration required for aggregation (Table 1). In addition, the lectin solution of this concentration was incubated with sugar of various kinds and various concentrations for 30 minutes, and then reacted with the red blood cell suspension again to observe the inhibition of aggregation by sugar (Table 2). As a result, agglutination reaction was observed at 8μg / mL level for both KML-C, KML-IIU and KML-IIL, and the aggregation effect was inhibited by Lactose, galactose and N-acetylgalactoseamine. Therefore, Korean lectins isolated from the present invention were identified as lectins having specipicity in Lactose, galactose, and N-acetylgalactoseamine.
실시예 6. KML-ⅡU와 KML-ⅡL에 대한 아미노산 서열 결정Example 6. Amino Acid Sequence Determination for KML-IIU and KML-IIL
각각의 시료에 대한 SDS-PAGE를 실시하고 PVDF membrane로 transfer 시킨후 각각의 chain에 대한 sequencing을 amino acid sequence에서 실시한 결과는 도 4와 같다.After performing SDS-PAGE on each sample and transferring to PVDF membrane, the sequencing of each chain in the amino acid sequence is shown in FIG. 4.
실시예 7. 한국산 겨우살이 lectin 유전자의 부분 클로닝Example 7. Partial Cloning of Korean Mistletoe lectin Gene
정제된 KML-ⅡU, KML-ⅡL에 대한 실시예 6에서 수행한 아미노산 서열을 기초로 하여, 2개의 올리고누클에오티드 프라이머를 설계하였다(Primer1: 5'-GTIACICATCAIACIGG-3', Primer2: 5'-ACIATICGCACIGTIGGTTC-3') 이 두 개의 프라이머를 이용하여 한국산 겨우살이의 전체 genomic DNA로부터 중합효소 연쇄반응(Polymerase Chain Reaction, PCR)을 실시하였다. CTAB방법으로 추출된 genomic DNA 1㎍, 각각의 primer 100pmol, 각각 200μM 농도의 dNTP, 1.5mM의 MgCl2, Amplitaq(Perkin-Elmer) DNA polymerase 2.5units로 조성된 PCR 반응액을 Thermal cycler(Perkin-Elmer 9600)에서 PCR을 수행하였다(denaturation at 94℃ 1min, annealing at 45℃ 2min, primer extension at 72℃ 2min, 35회 반복). PCR 수행결과 얻어진 약 800bp의 DNA를 Promega사에서 제조 판매하는 pGEM-T vector의 EcoR V위치(3'-overhang에 T가 붙어있어 PCR 산물의 클로닝에 적합함)에 연결시켜 클로닝 하였다. 재조합 플라스미드 벡터의 모식도는 도 5와 같다.Based on the amino acid sequence performed in Example 6 for purified KML-IIU, KML-IIL, two oligonucleotide primers were designed (Primer1: 5'-GTIACICATCAIACIGG-3 ', Primer2: 5'). -ACIATICGCACIGTIGGTTC-3 ') Polymerase Chain Reaction (PCR) was performed from the total genomic DNA of Korean mistletoe using these two primers. Thermal cycler (Perkin-Elmer) was prepared using 1 µg of genomic DNA extracted by CTAB method, 100 pmol of each primer, 200 μM of dNTP, 1.5 mM MgCl 2 , and Amplitaq (Perkin-Elmer) DNA polymerase 2.5units. 9600) was performed (denaturation at 94 ° C 1min, annealing at 45 ° C 2min, primer extension at 72 ° C 2min, repeated 35 times). About 800bp of DNA obtained as a result of PCR was cloned by connecting to EcoR V position (suitable for cloning of PCR product because T is attached to 3'-overhang) of pGEM-T vector manufactured by Promega. A schematic diagram of the recombinant plasmid vector is shown in FIG. 5.
실시예 8. 한국산 겨우살이 lectin 유전자의 부분염기서열과 추론된 아미노산 서열Example 8. Partial base sequence and deduced amino acid sequence of Korean mistletoe lectin gene
클로닝된 lectin 유전자의 클론중 2개를 DNA 염기서열을 automated sequencer를 이용하여 결정한 결과는 다음의 염기서열1과 염기서열2와 같다. 종래 식물의 lectin들은 대개 여러 가지 isoform이 함께 존재하는 것으로 많이 보고되어 있는데 한국산 겨우살이의 경우에도 sequencing 결과 클론간에 염기서열의 다소간의 차이를 보여 여러 isoform이 존재할 가능성이 시사되었다. 또한 DNA 염기서열로부터 아미노산 서열을 추론하여 기존에 알려진 유럽산 겨우살이 lectin 및 연관된 다른 식물의 lectin의 서열과 비교한 결과는 도 6과 같다.Two clones of the cloned lectin gene were determined using an automated sequencer for DNA sequencing, as shown in the following nucleotide sequence 1 and nucleotide sequence 2. Conventional plant lectins have been reported to have several isoforms together. In the case of Korean mistletoe, the sequencing results showed some differences in nucleotide sequences among the clones, suggesting the possibility of multiple isoforms. In addition, the amino acid sequence is deduced from the DNA base sequence and compared with the known European mistletoe lectin and the sequence of the lectin of other related plants as shown in FIG.
실시예 9. KML-ⅡU 및 KML-UUL의 암세포에 대한 세포독성 실험Example 9. Cytotoxicity Test of Cancer Cells of KML-IIU and KML-UUL
분리한 한국산 겨우살이 렉틴의 특성조사를 위해 유럽산 겨우살이 렉틴(EML-1)을 대조군으로 하여 다양한 세포주에 대한 세포독성 효과를 in vitro에서 관찰하였다. 먼저 각각의 세포주 일정량을 96 well plate에 넣고 다양한 농도의 렉틴을 첨가하고 48시간 후에 MTT법에 의한 세포의 성장을 조사하여 각 종양세포주의 성장을 50% 억제한 시료의 농도(ED50)를 표 3에 나타내었다. 실험결과, KML-ⅡU는 6-BL6 melanoma, Meth A Fibrosarcoma 세포주에서 비교적 높은 저항성을 나타낸 반면, 3LL carcinoma, Raji lymphoma에 대해서는 비교적 높은 감수성을 나타내어 종양세포에 대한 일부 특이성이 있는 것으로 생각되었다. 또한 대부분의 종양세포에 있어서 KML-ⅡU의 세포독성 효과는 유럽산 렉틴보다 강한 것으로 나타났으며 세포주에 따라서는 10배 이상 높은 세포독성 효과를 나타냈다. colon26-M3.1 carcinoma, B16-BL6 melanoma 및 L1210 leukemia 세포주에서 KML-ⅡU 및 KML-ⅡL의 세포독성 효과를 비교한 결과 모든 경우에서 KML-ⅡU가 비교적 높은 세포독성 효과를 나타냈다.For the characterization of isolated Korean mistletoe lectins, the cytotoxic effect on various cell lines was observed in vitro using European mistletoe lectin (EML-1) as a control. First, put a certain amount of each cell line into a 96 well plate, add various concentrations of lectin, and after 48 hours, examine the growth of cells by MTT method and measure the concentration of the sample that inhibited the growth of each tumor cell line by 50% (ED 50 ). 3 is shown. As a result, KML-IIU showed relatively high resistance to 6-BL6 melanoma and Meth A Fibrosarcoma cell lines, while it showed relatively high sensitivity to 3LL carcinoma and Raji lymphoma. In addition, the cytotoxic effect of KML-IIU was stronger than that of European lectin in most tumor cells, and the cell line showed more than 10 times higher cytotoxic effect. Comparing the cytotoxic effects of KML-IIU and KML-IIL in colon26-M3.1 carcinoma, B16-BL6 melanoma and L1210 leukemia cell lines, KML-IIU showed relatively high cytotoxic effects in all cases.
실시예 10. 면역관련 세포의 mitogen에 대한 반응성Example 10. Reactivity of mitogen to immune related cells
6주령의 Balb/c(암컷) 마우스 3마리를 한 group으로 하여 50ng의 KML-ⅡU를 주사(i.v.)하고 2일 후에 비장세포를 분리하였다. Flatbottoned 96-well culture plate(Nunc, Denmark)에 각 비장세포를 5×105/100㎕의 밀도를 각 well에 넣고, T 세포 및 B 세포의 mitogen으로 알려진 Con. A와 LPS를 각각 0.5 및 5㎍/㎖의 농도로 100㎕씩 각 well에 처리후 5% CO2, 37℃의 조건에서 3일간 배양하였다. 배양종료 6시간 전에 0.5μCi의 [3H]-TdR을 50㎕/well로 첨가후, 배양세포를 Filter mate 196(Packard Instrument, Meriden, CT)에 의해 glass filter에 흡착시킨 뒤, Matrix 96TMDirect Beta Counter(Packard)에서 방사선 활성을 측정하였다. 도 9에서 나타낸 바와같이 KML-ⅡU을 투여한 마우스의 비장세포는 mitogen을 첨가하지 않은 경우에서도 시료 무처리 대조군에 비하여 DNA의 합성이 증가되는 결과를 나타내어 각 시료의 투여가 마우스의 비장세포를 in vitro에서 자극한다는 것이 확인되었고 이때, mitogen을 첨가할 경우 mitogen의 반응성을 증가시키는 것으로 사료되었으며 그 반응성은 KML-ⅡU의 농도에 비례하는 결과를 나타내었다. 이상의 실험결과를 종합하여 볼 때 KML-ⅡU은 성숙된 숙주의 면역담당세포들이 효과적인 면역반응을 위한 일련의 세포적 반응성이 증진시키는 효과를 가지고 있었으며, 만약 외부로부터의 항원에 노출되었을 경우 유도되는 항원특이적 면역반응을 위한 작동세포의 수를 증폭시킴으로 항원에 대한 효과적인 면역반응이 유도될 것으로 사료되었다.Three 6-week-old Balb / c (female) mice were injected (iv) with 50 ng of KML-IIU as a group, and splenocytes were separated 2 days later. Flatbottoned 96-well culture plate into the respective cells in the spleen (Nunc, Denmark) a density of 5 × 10 5 / 100㎕ each well, a known mitogen for T cells and B cells Con. A and LPS were incubated in each well for 100 μl at concentrations of 0.5 and 5 μg / ml, respectively, and then incubated at 5% CO 2 and 37 ° C. for 3 days. After 6 hours of incubation, 0.5 μCi of [ 3 H] -TdR was added to 50 μl / well, and the cultured cells were adsorbed onto the glass filter by Filter mate 196 (Packard Instrument, Meriden, CT), followed by Matrix 96 TM Direct. Radioactivity was measured at Beta Counter (Packard). As shown in FIG. 9, splenocytes of mice administered with KML-IIU showed increased DNA synthesis compared to untreated samples even when mitogen was not added. It was found to be stimulated in vitro, and the addition of mitogen was thought to increase the reactivity of mitogen, which was proportional to the concentration of KML-IIU. Taken together, KML-IIU has the effect of enhancing the cellular reactivity of mature host immune cells for an effective immune response. By amplifying the number of effector cells for a specific immune response, an effective immune response to the antigen was induced.
실시예 11. 대식세포로부터 cytokines의 유도Example 11 Induction of Cytokines from Macrophages
Balb/c 마우스에 1% thioglycollate를 1㎖ 복강주사하고 4일 후에 경추탈골 방법으로 마우스를 희생시킨 후, 복강에 RPMI 1640 배지 10㎖를 주입하고 복벽을 가볍게 두드려 복강내 세포를 잘 섞이게 한 후 복강내세포(peritoneal exaudative cells; PEC)를 수집하였다. 수확한 PEC을 24 well culture plate에 1.5×106의 농도로 조정하여 plating하였다. 2시간 동안 배양하여 대식세포를 부착 후, 배양액으로 세척하여 부착되지 않은 세포를 제거하고, 시료로서 여러농도의 KML-ⅡU을 첨가하여 대식세포를 자극하였다. IL-1의 활성은 ELISA kit(ENDOGEN)를 이용하여 cytokine(IL-1)의 분비유도를 확인하였으며, TNF-α의 활성은 L929세포를 이용하는 생물학적 정량에 의하여 조사하였다. 이때, 시료의 L929세포에 대한 직접적인 영향을 배제하기 위하여 1시간 동안 대식세포를 자극후에 배양배지를 이용하여 세포를 4회 세척후, 새로운 배지를 넣고 24시간 배양하여 배양상등액을 준비하였다. TNF-α의 활성은 대식세포 배양상등액에 의한 L929세포의 증식억제로 조사하였으며, TNF-α의 분비 유도 확인은 rabbit-anti-TNF-α로서 TNF-α의 중화실험으로 실시하였다. 실험결과는 도 8에 나타내었으며 대식세포의 KML-ⅡU에 의한 IL-1 유도활성은 KML-ⅡU의 농도 50ng/mL부터 1ng/mL까지 유의하게 나타났고 KML-ⅡU 농도에 의존적인 경향을 나타냈다. TNF-α의 활성의 조사결과, KML-ⅡU으로 자극된 대식세포 배양상등액은 지극한 시료의 농도에 의존적으로 L929 세포주를 살해하였으며, 그 세포독성 효과는 rabbit anti-TNF-α에 의하여 억제됨으로서 KML-ⅡU은 대식세포를 자극하여 TNF-α를 유도하는 능력이 있는 것으로 사료되었다. In vitro에서 TNF-α를 유도하는 KML-ⅡU의 경우는 2~0.4ng/mL의 농도에서 유효하였다.4 ml of 1% thioglycollate was intraperitoneally injected in Balb / c mice, and after 4 days, the mice were sacrificed by the cervical dislocation method. Peritoneal exaudative cells (PEC) were collected. The harvested PEC was plated by adjusting the concentration to 1.5 × 10 6 on a 24 well culture plate. After culturing for 2 hours to attach macrophages, the cells were washed with culture to remove non-attached cells, and then, various concentrations of KML-IIU were added as samples to stimulate macrophages. IL-1 activity was confirmed by ELISA kit (ENDOGEN) induction of secretion of cytokine (IL-1), TNF-α activity was investigated by biological quantification using L929 cells. At this time, after stimulating the macrophages for 1 hour to exclude the direct effect on the sample L929 cells, the cells were washed four times using a culture medium, and then cultured for 24 hours to prepare a culture supernatant. TNF-α activity was examined by inhibition of proliferation of L929 cells by macrophage culture supernatant. TNF-α secretion was confirmed by rabbit-anti-TNF-α neutralization experiment. The experimental results are shown in FIG. 8, and IL-1 induced activity of macrophages by KML-IIU was significantly increased from 50ng / mL to 1ng / mL of KML-IIU, and was dependent on KML-IIU concentration. As a result of the investigation of TNF-α activity, KML-IIU-stimulated macrophage culture supernatants killed L929 cell line depending on the concentration of the sample, and its cytotoxic effect was inhibited by rabbit anti-TNF-α. -IIU was thought to be capable of inducing TNF-α by stimulating macrophages. KML-IIU, which induces TNF-α in vitro, was effective at a concentration of 2 ~ 0.4ng / mL.
실시예 12. NK-세포의 활성 측정Example 12 Measurement of Activity of NK-Cells
Balb/c 마우스에서 KML-ⅡU을 정맥주사하여 3일 후, 각 마우스의 비장세포와 YAC-1 세포의 비가 100:1, 50:1 및 25:1이 되도록 조종한 후 18시간 배양하였다. 배양종료 6시간전에 0.5μCi의 [3H]-TdR을 첨가후, 배양세포가 이용한 [3H]-TdR의 양을 Beta Counter를 이용하여 측정하였으며 NK 세포의 종양세포 살해능의 측정은 다음식에 의하여 구하였다.After 3 days of intravenous injection of KML-IIU in Balb / c mice, the mice were incubated for 18 hours after controlling the ratio of splenocytes and YAC-1 cells to 100: 1, 50: 1 and 25: 1. Six hours before the end of the culture, 0.5 [mu] Ci of [ 3 H] -TdR was added, and the amount of [ 3 H] -TdR used by the cultured cells was measured using the Beta Counter. Obtained by
Cytostatic activity(%)=(1-실험군의 YAC-1 세포의 총 [3H]-TdR uptake양/대조군의 YAC-1 세포의 총 [3H]-TdR uptake양)×100Cytostatic activity (%) = (YAC -1 cells in a total of 1-group [3 H] -TdR uptake amount / total of the YAC-1 cells in the control group [3 H] -TdR uptake quantity) × 100
실험결과는 도 9에 나타낸 것처럼 KML-ⅡU 50~10ng의 투여 사이에서 유효한 NK-세포의 종양세포 살해능을 나타내었다.Experimental results showed effective NK-cell tumor cell killing ability between 50 ~ 10ng KML-IIU administration as shown in FIG.
실시예 13. KML-ⅡU의 KLH 항원에 대한 항체유도의 증진효과Example 13. Effect of Enhancing Antibody Induction of KML-IIU to KLH Antigen
항원으로 KLH를 단독 혹은 KML-ⅡU와 동시에 면역한 후, KLH에 특이적인 항체의 유도능을 ELISA로 조사하였다. 도 10에 나타나 있듯이 KLH 항원 단독으로 투여한 경우보다 KML-ⅡU 20ng와 동시투여가 통계적으로 유의한 항체역가 상승효과가 일부 인정되었으나 booster 주사를 한 경우에는 그 효과가 현저하게 나타나 최초면역 5주째에는 KLH 단독투여의 항체역가에 비하여 60배 이상의 항체역가 상승효과가 있었으며 그 효과는 항원투여 10주째까지 유의성이 있었다. 따라서 KML-ⅡU은 항원에 대한 체액성 면역계를 활성화시키는 adjuvant 효과가 있었으며 이때 증진된 항체의 subisotype를 조사한 결과 도 10에 나타낸 바와 같이 주로 IgG1, IgG2a 및 IgG2b type이 상승된 결과를 나타내었다.After immunizing KLH alone or simultaneously with KML-IIU as an antigen, the inducibility of KLH-specific antibodies was examined by ELISA. As shown in FIG. 10, the synergistic effect of KL-IIU 20ng compared with the KLH antigen alone was statistically significant, but the booster injection resulted in marked effects. The antibody titer was 60 times higher than the antibody titer of KLH alone, and the effect was significant up to 10 weeks of challenge. Therefore, KML-IIU had an adjuvant effect of activating the humoral immune system to the antigen. As a result of investigating the subisotype of the enhanced antibody, as shown in FIG. 10, the IgG1, IgG2a and IgG2b types were elevated.
실시예 14. Lymphocyte 자극실험Example 14. Lymphocyte Stimulation Experiment
group당 3마리의 Balb/c 마우스에 20㎍의 KLH을 PBS에 용해한 후, KLH 단독 혹은 KML-ⅡU(20ng)을 혼합하여 2주 간격으로 총 2회 피하주사(100㎕/마우스)로 면역하였다. 최종 면역 2주 후에 면역한 마우스로 혹은 정상 마우스로부터 비장을 취하고 10% FBS가 함유된 RPMI-1640 배지에 현탁하여 세포의 농도가 1.5×105/ well이 되도록 조정한 후 96-well culture plate에 분주하였다. 비장세포가 분주된 각 well에 항원 KLH를 40㎍/㎖부터 4배씩 희석하여 0.039㎍/㎖의 농도로 조정하여 첨가하였으며 LPS 혹은 Con-A를 최종농도가 0.2㎍/㎖가 되도록 조정하여 넣고, 37℃, 5% CO2의 조건하에서 72시간 배양하였다. 배양종료 5시간 전에 0.5μCi의 [3H]-TdR(50㎕/well)을 각 well에 넣고 각 well의 방사선 활성을 측정하였다. 도 14에 나타낸 바와 같이 KML-ⅡU을 동시에 주사한 마우스의 비장세포는 KLH 단독 주사한 마우스의 비장세포에 비하여 항원으로 사용한 KLH에 대하여 유효한 세포의 분화(proliferation) 활성을 나타내었다. KLH에 대하여 감작된 비장세포의 분화 활성은 in vitro에서 재자극하는 KLH의 농도에 의존하는 경향을 나타냄으로서 KML-ⅡU의 in vitro에서의 항원에 대한 adjuvant 활성이 항원 특이적임을 나타냈다.Three Balb / c mice per group were lysed with 20 μg of KLH in PBS, followed by immunization with KLH alone or KML-IIU (20 ng) twice a total of 2 subcutaneous injections (100 μl / mouse) at 2 week intervals. . Two weeks after the last immunization, spleens were taken from immunized mice or from normal mice and suspended in RPMI-1640 medium containing 10% FBS to give a concentration of 1.5 × 10 cells.5Of The wells were adjusted and dispensed into 96-well culture plates. In each well of the splenocytes, antigen KLH was diluted 4 times from 40 µg / ml, adjusted to a concentration of 0.039 µg / ml, and LPS or Con-A was adjusted to a final concentration of 0.2 µg / ml. 37 ℃, 5% CO2Incubated for 72 hours under the conditions of. 0.5 μCi of [5 hours before the end of culture.3H] -TdR (50 μl / well) was added to each well and the radioactivity of each well was measured. As shown in FIG. 14, splenocytes of mice injected with KML-IIU simultaneously showed effective proliferation activity of cells against KLH used as antigen, compared to splenocytes of mice injected with KLH alone. The differentiation activity of splenocytes sensitized against KLH tends to be dependent on the concentration of KLH restimulating in vitro, indicating that the adjuvant activity of KML-IIU against antigen in vitro is antigen specific.
실시예 15. 항원 특이적인 Cytokines의 분비유도 효과Example 15 Secretion Induction Effect of Antigen-Specific Cytokines
KLH 항원에 대한 adjuvant로서의 KML-ⅡU의 효과는 주로 T helper cell subunits로부터 분비되는 cytokine과 관련이 있을 것으로 사료되어 KLH에 감작된 마우스의 비장세포 배양상등액을 이용하여 Th1 및 Th2 type로부터 분비되는 IL-2 및 IL-4의 분비 여부를 ELISA Kit를 이용하여 조사한 결과는 도 13과 같다. 대조군인 PBS 투여 마우스의 경우 두 cytokines의 경우 모두에서 검출한계 이하의 cytokine 활성을 나타내었으나, 항원 KLH를 단독으로 피하주사(s.c.)한 마우스의 비장세포를 in vitro에서 항원으로 재자극한 경우 유도되는 IL-2 및 IL-4는 각각 105 및 10 pg/mL의 수준으로 유효한 분비 효과가 있었다. KLH에 adjuvant로서 KML-ⅡU을 동시에 투여한 경우는 KLH 단독의 경우에 비하여 두 cytokine 모두 2배 이상의 유도활성을 나타내었다. 이 결과는 KM-110 혹은 KML-ⅡU는 Th1 또는 Th2의 어떠한 types의 CD+T세포에 모두 작용을 함으로서, 이후의 면역작용이 체액성 뿐만 아니라 세포성 면역능도 상승시킬 수 있을 것으로 사료되었다.The effect of KML-IIU as an adjuvant on KLH antigen was mainly related to cytokine secreted from T helper cell subunits, and IL- secreted from Th1 and Th2 types using splenocyte culture supernatants of mice sensitized to KLH. 2 and IL-4 secretion was investigated using the ELISA Kit results are as shown in FIG. PBS-treated mice, which were the control group, showed cytokine activity below the limit of detection in both cytokines, but were induced when the splenocytes of a mouse subcutaneously injected with antigen KLH were restimulated with antigen in vitro. IL-2 and IL-4 had an effective secretory effect at levels of 105 and 10 pg / mL, respectively. When KML-IIU was simultaneously administered to KLH as adjuvant, both cytokines showed more than two-fold induction activity compared to KLH alone. These results suggest that KM-110 or KML-IIU can act on any type of CD + T cells, either Th1 or Th2, so that subsequent immunization can increase not only humoral but also cellular immunity.
실시예 16. 지연성과민반응에 미치는 영향Example 16 Effect on Delayed Hypersensitivity Response
항원에 대한 지연성과민반응(delayed type hypersensitivity; DTH)은 항원에 대한 Keyhole lympet hemocyanine(KLH;20㎍) 단독 혹은 KML-ⅡU(20ng)을 2주 간격으로 총 2회 피하주사법으로 면역하였다. 최종면역 14주 후에 항원에 대하여 미리 감작된 숙주동물의 발바닥에 항원인 KLH(50㎍)을 피하주사한 후 1주일 동안 매일의 항원에 의하여 유도되는 항원주사부위의 팽창정도를 항원 무처리군에 대한 비율로서 지연성과민반응의 유도를 측정하였다. 도 14에 나타낸 바와같이 20ng의 KML-ⅡU가 동시투여된 마우스의 지연성과민반응은 항원 단독 주사의 마우스에 비하여 항원 재투여 2일후까지 유의한 증강효과가 있어 지연성과민반응을 증진시키는 면역증강효과를 유도하는 것이 인정되었다.Delayed type hypersensitivity (DTH) to the antigen was immunized with Keyhole lympet hemocyanine (KLH; 20 ㎍) alone or KML-IIU (20 ng) to the antigen twice a week. After 14 weeks of final immunization, the subcutaneous injection of KLH (50 μg), an antigen, was applied to the paw of a host animal previously sensitized to the antigen. Induction of delayed and sensitive responses was measured as a ratio. As shown in FIG. 14, delayed hypersensitivity reactions of mice co-administered with 20 ng of KML-IIU had a significant enhancement effect up to 2 days after antigen re-administration compared to mice injected with antigen alone, thereby enhancing delayed hypersensitivity reactions. It was recognized to induce an effect.
이상 실시예에서 설명한 바와 같이, 본 발명 한국산 겨우살이 식물로부터 분리한 두가지 렉틴 단백질은 유럽산 겨우살이로부터 분리한 렉틴 단백질들과는 아미노산서열 및 그 암호화 유전자의 염기서열에 차이가 존재하였으며, 항원-특이적 면역반응을 증강시키는 매우 뛰어난 효과가 있어 본 발명은 생물의약산업상 매우 유용한 발명인 것이다.As described in the above examples, the two lectin proteins isolated from the Korean mistletoe plants of the present invention differed from the lectin proteins isolated from the European mistletoe in the amino acid sequence and the nucleotide sequence of the coding gene. There is a very good effect of enhancing the present invention is a very useful invention in the biomedical industry.
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