KR20240110699A - Monoclonal antibody specifically binding to KCa3.1 channel protein and composition for diagnosis of vascular diseases containing it - Google Patents
Monoclonal antibody specifically binding to KCa3.1 channel protein and composition for diagnosis of vascular diseases containing it Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
- C12N5/12—Fused cells, e.g. hybridomas
- C12N5/16—Animal cells
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2510/00—Genetically modified cells
- C12N2510/02—Cells for production
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- G—PHYSICS
- G01—MEASURING; TESTING
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Abstract
본 발명은 혈관내피세포의 KCa3.1 채널 단백질에 특이적으로 결합하는 단일클론 항체와, 상기 단일클론 항체를 생산하는 하이브리도마 세포주, 상기 하이브리도마 세포주를 이용한 단일클론 항체의 제조방법, 그리고 상기 단일클론 항체를 포함하는 혈관질환 진단용 조성물에 관한 것이다.The present invention provides a monoclonal antibody that specifically binds to the KCa3.1 channel protein of vascular endothelial cells, a hybridoma cell line producing the monoclonal antibody, a method for producing a monoclonal antibody using the hybridoma cell line, and It relates to a composition for diagnosing vascular diseases containing the monoclonal antibody.
Description
본 발명은 혈관내피세포의 KCa3.1 채널 단백질에 특이적으로 결합하는 단일클론 항체와, 상기 단일클론 항체를 생산하는 하이브리도마 세포주, 상기 하이브리도마 세포주를 이용한 단일클론 항체의 제조방법, 그리고 상기 단일클론 항체를 포함하는 혈관질환 진단용 조성물에 관한 것이다.The present invention provides a monoclonal antibody that specifically binds to the KCa3.1 channel protein of vascular endothelial cells, a hybridoma cell line producing the monoclonal antibody, a method for producing a monoclonal antibody using the hybridoma cell line, and It relates to a composition for diagnosing vascular diseases containing the monoclonal antibody.
생체의 포타슘 채널(potassium channel)은 세포막에 존재하면서 세포의 안팎으로 포타슘 이온(K+)을 통과시키는 채널 단백질을 의미한다. 포타슘 채널 단백질은 세포 내 칼슘이온(Ca2+)의 농도와 흥분성을 조절하는 등 세포의 기능에 큰 영향을 미친다. 세포 내 칼슘이온에 의해 활성화되는 포타슘 채널에는 여러 종류가 있는데, 혈관내피세포에는 KCa2.3 및 KCa3.1 채널 등이 존재하며, 이중 KCa3.1 채널이 가장 중요한 역할을 하는 것으로 알려져 있다.The biological potassium channel refers to a channel protein that exists in the cell membrane and allows potassium ions (K + ) to pass into and out of the cell. Potassium channel proteins have a significant impact on cellular functions, such as regulating the concentration and excitability of intracellular calcium ions (Ca 2+ ). There are several types of potassium channels activated by intracellular calcium ions. In vascular endothelial cells, KCa2.3 and KCa3.1 channels exist, of which KCa3.1 channel is known to play the most important role.
혈관내피세포는 혈장과 직접 접촉하므로 혈장 속에 있는 각종 혈관질환 유발물질이 KCa3.1 채널의 발현을 감소시켜서 혈관내피세포의 기능부전을 유발한다(Choi Set al, 2013a, Free Radic Biol Med, 57, 10-21). 그리고 혈관내피세포의 기능부전은 각종 혈관질환을 일으키는 원인이 된다. 따라서 종래에도 KCa3.1 채널 단백질의 발현 수준을 바이오마커로 사용하여 혈관질환을 포함하는 각종 KCa3.1 채널 매개질환을 진단하는 기술들이 제시되어 있다.Since vascular endothelial cells are in direct contact with plasma, various vascular disease-causing substances in plasma reduce the expression of KCa3.1 channels, causing dysfunction of vascular endothelial cells (Choi Set al, 2013a, Free Radic Biol Med, 57, 10-21). And dysfunction of vascular endothelial cells causes various vascular diseases. Therefore, conventional technologies have been proposed to diagnose various KCa3.1 channel-mediated diseases, including vascular diseases, by using the expression level of the KCa3.1 channel protein as a biomarker.
예를 들면, 대한민국 특허 제10-1517689호(2015년 04월 28일)에는 KCa3.1 채널 단백질의 에피토프에 특이적으로 결합하는 항체 및 이를 이용한 KCa3.1 채널 매개질환의 진단용 조성물이 개시되어 있다. 여기서 상기 KCa3.1 채널 매개질환으로는 면역질환, 염증성 질환, 뇌질환, 암질환 등이 예시되어 있다.For example, Republic of Korea Patent No. 10-1517689 (April 28, 2015) discloses an antibody that specifically binds to an epitope of the KCa3.1 channel protein and a composition for diagnosing KCa3.1 channel-mediated diseases using the same. . Here, examples of the KCa3.1 channel-mediated diseases include immune diseases, inflammatory diseases, brain diseases, and cancer diseases.
또한 특허 제10-1889594호(2018년 08월 10일) 및 특허 제10-1903230호(2018년 09월 20일)에는, Rab5C, 클라트린(clathrin), 카베올린-1(caveolin-1), EEA1(Early Endosome Antigen 1), 그리고 KCa2.3 또는 KCa3.1 채널 단백질의 발현 수준을 측정함으로써, 혈관내피세포의 기능부전을 진단할 수 있는 혈관질환 진단용 조성물이 개시되어 있다.Additionally, in Patent No. 10-1889594 (August 10, 2018) and Patent No. 10-1903230 (September 20, 2018), Rab5C, clathrin, caveolin-1, A composition for diagnosing vascular diseases that can diagnose dysfunction of vascular endothelial cells by measuring the expression levels of EEA1 (Early Endosome Antigen 1) and KCa2.3 or KCa3.1 channel proteins is disclosed.
그 외에도, 특허 제10-2047502호(2019년 11월 15일) 및 이에 대응하는 국제공개 WO2017/222221(2017년 12월 28일)에는, KCa2.3, KCa3.1 채널 단백질, 또는 이들 단백질을 코딩하는 유전자로부터 발현된 mRNA의 발현 수준을 암의 발병이 의심되는 개체의 혈청에 노출시킨 혈관내피세포에서 측정할 수 있는 제제를 포함하는, 암 진단용 조성물이 개시되어 있다.In addition, in Patent No. 10-2047502 (November 15, 2019) and the corresponding international publication WO2017/222221 (December 28, 2017), KCa2.3, KCa3.1 channel proteins, or these proteins A composition for diagnosing cancer is disclosed, which includes an agent capable of measuring the expression level of mRNA expressed from a coding gene in vascular endothelial cells exposed to the serum of an individual suspected of having cancer.
이상 살핀 바와 같이, KCa3.1 채널 매개질환 중에서 특히 혈관질환을 조기에 진단할 수 있는 바이오마커로서 KCa3.1 채널 단백질에 특이적으로 결합하는 항체가 유용하게 사용될 수 있다. As discussed above, an antibody that specifically binds to the KCa3.1 channel protein can be usefully used as a biomarker for early diagnosis of vascular diseases, especially among KCa3.1 channel-mediated diseases.
그런데 종래에 개발된 KCa3.1 채널 항체는 상기 특허 제10-1517689호(2015년 04월 28일)에서 보는 바와 같이, 화학적으로 합성된 특정 아미노산 서열을 갖는 면역원을 사용하여 토끼를 통해서 제작한 다클론 항체(polyclonal antibody)로서, 특정 에피토프를 표적으로 하는 특이성이 떨어지고, 동일한 다클론 항체의 대량 생산이 어려워서 이를 산업적인 규모에 적용하기는 부족한 측면이 있었다. However, as shown in Patent No. 10-1517689 (April 28, 2015), the previously developed KCa3.1 channel antibody is produced in rabbits using a chemically synthesized immunogen with a specific amino acid sequence. As a polyclonal antibody, it has poor specificity for targeting a specific epitope and it is difficult to mass-produce the same polyclonal antibody, making it difficult to apply it on an industrial scale.
이에 본 발명의 목적은, 인체의 KCa3.1 채널 단백질에 특이적으로 결합하고, 재현성이 우수하며, 산업적인 규모에서 안정적인 공급이 가능한 신규의 단일클론 항체 및 그 제조방법을 제공하는 것이다.Accordingly, the purpose of the present invention is to provide a novel monoclonal antibody that specifically binds to the human KCa3.1 channel protein, has excellent reproducibility, and can be supplied stably on an industrial scale, and a method for producing the same.
본 발명의 다른 목적은, 인체의 KCa3.1 채널 단백질에 특이적으로 결합하는 단일클론 항체를 생산하는 신규의 하이브로도마 세포주를 제공하는 것이다.Another object of the present invention is to provide a novel hybridoma cell line that produces a monoclonal antibody that specifically binds to the human KCa3.1 channel protein.
본 발명의 또 다른 목적은, KCa3.1 채널 단백질의 발현 수준을 바이오마커로 하여 KCa3.1 채널 매개질환, 특히 혈관질환을 조기에 진단할 수 있는 진단용 조성물을 제공하는 것이다.Another object of the present invention is to provide a diagnostic composition that can diagnose KCa3.1 channel-mediated diseases, especially vascular diseases, at an early stage by using the expression level of the KCa3.1 channel protein as a biomarker.
본 발명에 따른 신규의 단일클론 항체는, 수탁번호 KCTC 15261BP인 하이브리도마 3E1 세포주 또는 수탁번호 KCTC 15262BP인 하이브리도마 4B9 세포주에 의해 생산되고, 인체의 KCa3.1 채널 단백질에 특이적으로 결합하는 것을 특징으로 한다.The novel monoclonal antibody according to the present invention is produced by the hybridoma 3E1 cell line with accession number KCTC 15261BP or the hybridoma 4B9 cell line with accession number KCTC 15262BP, and binds specifically to the human KCa3.1 channel protein. It is characterized by
본 발명에 따른 신규의 하이브리도마 세포주는, 인체의 KCa3.1 채널 단백질에 특이적으로 결합하는 단일클론 항체를 생산하는 것으로, 수탁번호 KCTC 15261BP인 하이브리도마 3E1 세포주 수탁번호 KCTC 15262BP인 하이브리도마 4B9 세포주이다. 상기 3E1 세포주와 4B9 세포주는 2022년 12월 26일자에 기탁되었다.The new hybridoma cell line according to the present invention produces a monoclonal antibody that specifically binds to the human KCa3.1 channel protein, and is a hybridoma 3E1 cell line with accession number KCTC 15261BP and a hybrid with accession number KCTC 15262BP. Chopping board 4B9 cell line. The 3E1 cell line and 4B9 cell line were deposited on December 26, 2022.
본 발명에 따른 단일클론 항체의 제조방법은, A) pcDNA3.1 발현벡터에다 KCa3.1 채널 단백질의 유전자를 삽입하여 재조합 벡터(pcDNA3.1-KCa3.1)를 제조하는 단계와; B) 상기 재조합 벡터(pcDNA3.1-KCa3.1)를 발현시킨 ExpiCHO 세포주에서 재조합 KCa3.1 채널 단백질을 분리 및 정제하는 단계와; C) 상기 재조합 KCa3.1 채널 단백질을 투입하여 면역을 유도한 마우스의 비장에서 B 림프구를 분리하고, 이를 골수종 세포와 혼합하여 하이브리도마를 제조하는 단계와; D) 상기 하이브리도마 중에서 KCa3.1 채널 단백질에 특이적으로 반응하는 세포주를 클로닝하여 수탁번호 KCTC 15261BP인 3E1 세포주와 수탁번호 KCTC 15262BP인 4B9 세포주를 확보하는 단계와; E) 상기 하이브리도마 3E1 세포주 또는 4B9 세포주를 배양하여 단일클론 항체를 수득하는 단계; 를 포함하는 것을 특징으로 한다.The method for producing a monoclonal antibody according to the present invention includes the steps of A) preparing a recombinant vector (pcDNA3.1-KCa3.1) by inserting the gene for the KCa3.1 channel protein into the pcDNA3.1 expression vector; B) isolating and purifying the recombinant KCa3.1 channel protein from the ExpiCHO cell line expressing the recombinant vector (pcDNA3.1-KCa3.1); C) isolating B lymphocytes from the spleen of mice immunized with the recombinant KCa3.1 channel protein and mixing them with myeloma cells to prepare hybridomas; D) cloning a cell line that specifically responds to the KCa3.1 channel protein among the hybridomas to obtain a 3E1 cell line with accession number KCTC 15261BP and a 4B9 cell line with accession number KCTC 15262BP; E) culturing the hybridoma 3E1 cell line or 4B9 cell line to obtain a monoclonal antibody; It is characterized by including.
본 발명에서 상기 KCa3.1 채널 단백질의 서열정보는 미국 국립생물정보센터(National Center for Biotechnology Information; NCBI) 등과 같은 공지의 데이터베이스로부터 획득할 수 있다. 예를 들어, 상기 KCa3.1 채널 단백질은 NCBI GenBank Acession NO. NM_002250, NM_001163510, NP_002241 또는 NP_001156982일 수 있으나, 이에 제한되지 않는다.In the present invention, sequence information of the KCa3.1 channel protein can be obtained from known databases such as the U.S. National Center for Biotechnology Information (NCBI). For example, the KCa3.1 channel protein is described in NCBI GenBank Acession NO. It may be NM_002250, NM_001163510, NP_002241 or NP_001156982, but is not limited thereto.
마지막으로 본 발명에 따른 혈관진단용 조성물은, 인체의 KCa3.1 채널 단백질의 발현 수준을 측정하는 바이오마커로서, 수탁번호 KCTC 15261BP인 하이브리도마 3E1 세포주 또는 수탁번호 KCTC15262BP인 하이브리도마 4B9 세포주에 의해 생산되는 단일클론 항체를 포함하는 것을 특징으로 한다.Lastly, the composition for vascular diagnosis according to the present invention is a biomarker that measures the expression level of the KCa3.1 channel protein in the human body, and is measured by the hybridoma 3E1 cell line with accession number KCTC 15261BP or the hybridoma 4B9 cell line with accession number KCTC15262BP. It is characterized in that it contains a monoclonal antibody produced.
상기 혈관진단용 조성물을 사용하여 시험대상 개체로부터 분리된 생물학적 시료에 대한 KCa3.1 단백질의 발현 수준을 측정하고, 그 결과를 정상 개체로부터 분리된 생물학적 시료에 대한 측정값과 비교한 다음, 시험대상 개체의 측정값이 더 높으면 혈관질환이 있는 것으로 판단할 수 있다.Using the composition for vascular diagnosis, the expression level of KCa3.1 protein is measured on a biological sample isolated from a test subject, the results are compared with the measured value on a biological sample isolated from a normal subject, and then the test subject If the measurement value is higher, it can be determined that there is vascular disease.
본 발명에 따르면, 인체의 KCa3.1 채널 단백질에 특이적으로 결합하는 단일클론 항체를 균일한 품질로 대량생산 제조할 수 있고, 상기 단일클론 항체를 사용하면, 각종 KCa3.1 채널 매개질환, 특히 혈관질환을 조기에 진단할 수 있는 효과가 있다. According to the present invention, a monoclonal antibody that specifically binds to the human KCa3.1 channel protein can be mass-produced with uniform quality, and the use of the monoclonal antibody can cause various KCa3.1 channel-mediated diseases, especially It is effective in diagnosing vascular diseases early.
또한, 본 발명에 따른 단일클론 항체는 산업적으로 혈관질환 진단용 조성물은 물론, 혈관질환용 진단키트를 제조하는 바이오마커로도 유용하게 사용될 수 있을 것으로 기대된다.In addition, the monoclonal antibody according to the present invention is expected to be useful industrially as a composition for diagnosing vascular diseases, as well as a biomarker for manufacturing diagnostic kits for vascular diseases.
도 1은 본 발명에 따른 재조합 벡터(pcDNA3.1-KCa3.1)의 벡터 맵이고,
도 2는 재조합 KCa3.1 채널 단백질을 대량 배양한 후 SDS-PAGE를 통해서 분리 정제된 단백질의 크기를 확인한 결과를 나타낸 것이며,
도 3은 SDS-PAGE를 통해서 본 발명에 따른 단일클론 항체를 분리 및 정제한 결과를 나타낸 것이다.Figure 1 is a vector map of the recombinant vector (pcDNA3.1-KCa3.1) according to the present invention,
Figure 2 shows the results of mass culturing the recombinant KCa3.1 channel protein and then confirming the size of the separated and purified protein through SDS-PAGE.
Figure 3 shows the results of separation and purification of the monoclonal antibody according to the present invention through SDS-PAGE.
이하, 바람직한 실시예를 통하여 본 발명을 상세하게 설명한다. 그러나 하기 실시예에 의해서 본 발명의 권리범위가 제한되는 것은 아니다. 또한 본 발명을 실시 하는데 꼭 필요한 구성이라 하더라도 종래기술에 소개되어 있거나, 통상의 기술자가 공지기술로부터 용이하게 실시할 수 있는 사항에 대해서는 구체적인 설명을 생략한다. Hereinafter, the present invention will be described in detail through preferred embodiments. However, the scope of the present invention is not limited by the following examples. In addition, even if the configuration is essential for carrying out the present invention, detailed description will be omitted for matters that are introduced in the prior art or that can be easily implemented by a person skilled in the art from the known technology.
[ 실시예 1 ] 재조합 인간 KCa3.1 채널 단백질의 발현 [Example 1] Expression of recombinant human KCa3.1 channel protein
(1) KCa3.1 채널 단백질 발현을 위한 재조합 벡터의 제조(1) Preparation of recombinant vector for KCa3.1 channel protein expression
항원으로 사용할 인간 KCa3.1 채널 단백질을 PCR 방법으로 증폭하였다. pcDNA3.1(+) 발현벡터의 BamHI, EcoRI의 제한효소 자리에다 증폭된 KCa3.1 채널 단백질의 유전자(hKCNN4)를 삽입하여 재조합 벡터(pcDNA3.1-KCa3.1)를 제조하였다.The human KCa3.1 channel protein to be used as an antigen was amplified by PCR. A recombinant vector (pcDNA3.1-KCa3.1) was prepared by inserting the amplified KCa3.1 channel protein gene (hKCNN4) into the BamHI and EcoRI restriction enzyme sites of the pcDNA3.1(+) expression vector.
첨부 도 1은 앱클론(AbClon)사에 의뢰하여 제작한 재조합 플라스미드 벡터(pcDNA3.1-KCa3.1)의 벡터 맵을 나타낸 것이고, 서열목록 1은 상기 벡터에 도입된 인간 KCa3.1 채널 단백질 유전자(hKCNN4)의 염기서열을 나타낸 것이다. Attached Figure 1 shows a vector map of the recombinant plasmid vector (pcDNA3.1-KCa3.1) produced by requesting AbClon, and Sequence Listing 1 shows the human KCa3.1 channel protein gene introduced into the vector. This shows the base sequence of (hKCNN4).
(2) 재조합 KCa3.1 채널 단백질의 발현, 분리 및 정제(2) Expression, isolation and purification of recombinant KCa3.1 channel protein
FBS(fetal bovine serum)를 10% 포함한 RPMI1640 배지에서 ExpiCHO 세포주를 배양하고, ExpiFectamine(Thermo Scientific Fisher)을 이용하여 앱클론사의 지침에 따라 재조합 벡터(pcDNA3.1-KCa3.1)의 형질을 도입하였다. 상기 ExpiCHO 세포주를 배양하여 얻어진 세포 상층액을 여과한 후 1X PBS로 희석하였다. 준비된 단백질을 protein A resin에 결합시키고, 1X PBS로 세척 하였다. The ExpiCHO cell line was cultured in RPMI1640 medium containing 10% FBS (fetal bovine serum), and the recombinant vector (pcDNA3.1-KCa3.1) was introduced using ExpiFectamine (Thermo Scientific Fisher) according to Abclone's instructions. . The cell supernatant obtained by culturing the ExpiCHO cell line was filtered and diluted with 1X PBS. The prepared protein was bound to protein A resin and washed with 1X PBS.
pH 3.5인 0.1M 글라이신 버퍼로 단백질을 용출(elution)하고, pH 9.0 Tris 버퍼로 중성화하였다. 정제된 단백질을 알맞은 농도로 농축 및 투석하고, SDS-PAGE를 통해 확인하였다. 첨부 도 2는 SDS-PAGE 분석 결과를 나타낸 것으로, 42kDa 부근에 형성된 밴드에 의해서 재조합 KCa3.1 채널 단백질이 분리 및 정제된 것을 확인할 수 있다. Proteins were eluted with 0.1M glycine buffer at pH 3.5 and neutralized with Tris buffer at pH 9.0. The purified protein was concentrated and dialyzed to an appropriate concentration and confirmed through SDS-PAGE. Attached Figure 2 shows the results of SDS-PAGE analysis, and it can be confirmed that the recombinant KCa3.1 channel protein was separated and purified by the band formed around 42 kDa.
[ 실시예 2 ] 단일클론 항체를 생산하는 세포주의 클로닝[Example 2] Cloning of cell lines producing monoclonal antibodies
(1) 마우스에 대한 면역반응 유도 및 항체의 항원성 확인(1) Induction of immune response in mice and confirmation of antigenicity of antibodies
상기 실시예 1에서 제조한 재조합 KCa3.1 채널 단백질을 프로인트 완전 어주반트(complete Freund's adjuvant, Sigma)와 혼합하여 에멀젼을 제조하였다. 상기 에멀젼을 생후 6주령의 암컷 BALB/C 마우스의 복강 내에 1차 주사하고, 2주 후에 동일한 방법으로 2차 주사하였다. 2차의 주입 후 혈액 테스트를 통해 면역반응을 강하게 보이는 마우스에 대해 3차 주사(12주, IP or tail vein)를 수행하였다. 이러한 과정으로 면역화된 마우스의 혈액을 채혈한 후, ELISA법(450nm)으로 KCa3.1 채널 항체의 항원성 여부를 분석하였다. An emulsion was prepared by mixing the recombinant KCa3.1 channel protein prepared in Example 1 with complete Freund's adjuvant (Sigma). The emulsion was first injected into the abdominal cavity of a 6-week-old female BALB/C mouse, and 2 weeks later, it was injected a second time using the same method. After the second injection, a third injection (12 weeks, IP or tail vein) was performed on mice that showed a strong immune response through a blood test. After collecting blood from mice immunized through this process, the antigenicity of the KCa3.1 channel antibody was analyzed using ELISA (450 nm).
구체적으로, ELISA 확인용 항원(KCa3.1 채널 단백질)을 코팅 버퍼에 5㎍/㎖ 농도로 희석하고, 96 웰 플레이트에 한웰당 50㎕씩 분주 코팅한 후 4℃에서 하룻밤 동안 반응시켰다. 다음날 2% 탈지유로 블로킹하고, 1차 항체를 배율에 맞게 희석한 후, 플레이트 웰에 50㎕씩 분주하여 37℃에서 반응시켰다. Specifically, the antigen for ELISA confirmation (KCa3.1 channel protein) was diluted in coating buffer to a concentration of 5㎍/㎖, and 50㎕ per well was dispensed and coated on a 96-well plate, followed by reaction at 4°C overnight. The next day, the cells were blocked with 2% skim milk, the primary antibody was diluted according to the ratio, and 50 μl was dispensed into each well of the plate and reacted at 37°C.
반응이 완료된 플레이트는 TBS-T로 3회 세척하고, HRP가 접합되어 있는 Goat anti-mouse IgG를 1:10,000 비율로 희석한 후 플레이트 웰에 50㎕씩 분주하여 37℃에서 반응하였다. 반응 완료 후 세척하고, TMB 용액을 플레이트 웰에 50㎕씩 분주하여 5분 반응시킨 다음, 1N H2SO4 100㎕/well을 넣어 반응을 종결하였다. 마이크로플레이트 리더(Microplate reader)로 450nm에서 흡광도 값을 측정하여 세럼 내 타이터(titer)를 확인하였다.After the reaction was completed, the plate was washed three times with TBS-T, and goat anti-mouse IgG conjugated with HRP was diluted at a ratio of 1:10,000, and then 50 μl was dispensed into each well of the plate and reacted at 37°C. After completing the reaction, the plate was washed, 50 ㎕ of TMB solution was dispensed into each well of the plate and allowed to react for 5 minutes, and then 100 ㎕/well of 1N H2SO4 was added to terminate the reaction. The titer in the serum was confirmed by measuring the absorbance value at 450 nm with a microplate reader.
(2) 세포 융합 : 하이브리도마 세포주의 제조(2) Cell fusion: Preparation of hybridoma cell lines
상기 세럼 내 타이터를 기준으로 높은 항체 역가가 높은 면역 동물을 선정하여 림프구를 분리하고, 세포융합을 실시하였다. 구체적으로, BALB/c 마우스 1마리당 항원 100μg을 어주반트와 섞어서 앱클론사의 MANI법(Monoclonal Antibody Next Innovation)을 이용하여 주사하였다. Based on the titer in the serum, immune animals with high antibody titers were selected, lymphocytes were isolated, and cell fusion was performed. Specifically, 100 μg of antigen per BALB/c mouse was mixed with adjuvant and injected using AbClon's MANI method (Monoclonal Antibody Next Innovation).
융합 3일 전 항원 100μg을 1X PBS에 섞어 주사하여 면역반응을 증가시키고, 3일 뒤 마우스의 비장에서 B 림프구를 분리하여 골수종 세포인 SP2/0-Ag14 (ATCC, CRL-1581)와 혼합한 다음, PEG-1500(Roche, 783641) 용액을 이용하여 융합시켰다. 융합된 세포는 하이포잔틴(hypoxanthin), 아미노프테린(aminopterine), 티미딘(thymidine)이 첨가된 배지(HAT supplement, Sigma-Aldrich, H0262)에 배양하여 골수종 세포와 B 림프구가 융합된 하이브리도마를 선택적으로 선별 배양하였다. HAT 배지의 선택으로 형질 전환된 하이브리도마만 살아남았다.3 days before fusion, 100 μg of antigen mixed in 1 , fusion was performed using PEG-1500 (Roche, 783641) solution. The fused cells were cultured in medium (HAT supplement, Sigma-Aldrich, H0262) supplemented with hypoxanthin, aminopterine, and thymidine to form a hybridoma in which myeloma cells and B lymphocytes were fused. were selectively cultured. Only transformed hybridomas survived selection on HAT medium.
(3) 하이브리도마 세포주의 클로닝(3) Cloning of hybridoma cell lines
96웰 플레이트에다 PKA 항원 100ng을 코팅버퍼(0.1M sodium carbonate buffer) 100μl에 희석하고, 로딩 후 37℃에서 배양하였다. 여기에 1X PBST 용액을 300μl/well씩 넣어 3차례 세척하고, 2% 탈지유 200μl로 실온에서 블록킹한 후, 1X PBST 용액을 300μl/well씩 넣어 3차례 세척하였다. 이어서, 하이브리도마 세포배양 상층액(sup.; 포획항체 포함액)을 100μl/well 씩 로딩하여 37℃에서 배양하였다. 앞의 과정과 같은 방법으로 다시 세척하고, 2차 항체로 항-마우스 IgG-HRP(Abclon, Abc5001)을 1:20,000로 희석하여 100μl/well씩 로딩한 후 37℃에서 배양하였다. 100ng of PKA antigen was diluted in 100μl of coating buffer (0.1M sodium carbonate buffer) in a 96-well plate, and cultured at 37°C after loading. It was washed three times by adding 300 μl/well of 1X PBST solution, blocked at room temperature with 200 μl of 2% skim milk, and washed three times by adding 300 μl/well of 1 Next, 100 μl/well of hybridoma cell culture supernatant (sup.; solution containing capture antibody) was loaded and cultured at 37°C. Washed again in the same manner as before, anti-mouse IgG-HRP (Abclon, Abc5001) was diluted 1:20,000 as a secondary antibody, loaded at 100 μl/well, and incubated at 37°C.
앞의 과정과 같은 방법으로 다시 세척하고, TMB 용액을 100μl/well씩 로딩한 후 발색하고, 1N 황산을 100μl/well씩 로딩하여 반응을 종료시킨 다음, 450nm에서 흡광도를 측정하였다. 이때 ELISA를 통해, 항원과 특이적으로 반응하는 양성세포는 연속 희석법(serial dilution method)을 이용하여 양성세포와 음성세포를 분리하는 클로닝을 반복하였다.Washed again in the same manner as the previous process, 100 μl/well of TMB solution was loaded for color development, 1N sulfuric acid was loaded at 100 μl/well to terminate the reaction, and the absorbance was measured at 450 nm. At this time, through ELISA, positive cells that specifically reacted with the antigen were repeatedly cloned to separate positive and negative cells using a serial dilution method.
다음 [표 1] 및 [표 2]와 같은 퓨전 스크리닝(fusion screening)을 거쳐서 항원에 특이적으로 반응하는 항체를 생산하는 하이브리도마 세포주를 선별하였다. 생산된 하이브리도마 세포는 각각 1 x 106cell/ml씩 동결하여 액체 질소통 안에 보관하였다. 하기 도표에서 ‘hKCNN4-hFc’는 Fc tagging된 KCa3.1 채널 단백질을 의미하고 ‘hFc control’은 hFc에 결합하는 항체를 배제하기 위해 사용한 대조군을 의미한다.Hybridoma cell lines producing antibodies that specifically react to antigens were selected through fusion screening as shown in [Table 1] and [Table 2]. The hybridoma cells produced were frozen at 1 x 10 6 cell/ml each and stored in a liquid nitrogen tank. In the diagram below, 'hKCNN4-hFc' refers to the Fc tagged KCa3.1 channel protein and 'hFc control' refers to the control used to exclude antibodies binding to hFc.
그 결과, KCa3.1 채널 단백질에 대하여 특이적인 단일클론 항체를 생산하는 하이브리도마 세포주 2종을 선별하였다. 이들을 각각 ‘3E1 세포주’ 및 ‘4B9 세포주’로 명명하고, 2022년 12월 26일자로 국제기탁기관인 한국생명공학연구원 생물자원센터(Korean Collection for Type Cultures; KCTC)에 기탁하였다. As a result, two hybridoma cell lines producing specific monoclonal antibodies against the KCa3.1 channel protein were selected. They were named ‘3E1 cell line’ and ‘4B9 cell line’, respectively, and were deposited with the Korea Collection for Type Cultures (KCTC), an international depository institution, on December 26, 2022.
(4) 단일클론 항체의 이소타입(isotype) 결정(4) Determination of isotype of monoclonal antibody
본 발명에 따른 단일클론 항체의 이소타입 결정을 위하여 상기 하이브리도마 3E1 세포주와 4B9 세포주로부터 얻은 배양액의 상층액을 이용하여 ELISA를 실시하였다. 구체적으로, 코팅 버퍼에 항원을 각 웰에 분주하여 코팅하였다. 상기 코팅 용액을 버리고, 1% 탈지유를 200㎕씩 각 웰에 분주하여 37℃에서 30분간 블록킹하였다. TBS-T로 1회 세척한 후, 상기 하이브리도마 세포주의 배양액을 해당 웰에 넣고 37℃에서 2시간 동안 반응시켰다. To determine the isotype of the monoclonal antibody according to the present invention, ELISA was performed using the culture supernatant obtained from the hybridoma 3E1 cell line and the 4B9 cell line. Specifically, the antigen was dispensed into each well in coating buffer and coated. The coating solution was discarded, and 200 ㎕ of 1% skim milk was dispensed into each well and blocked at 37°C for 30 minutes. After washing once with TBS-T, the culture medium of the hybridoma cell line was added to the corresponding well and reacted at 37°C for 2 hours.
TBS-T로 3회 세척하고, IgM, IgG1, IgG2a, IgG2b, IgG3, IgA, Kappa, Lambda를 각각 시험할 웰에 넣은 다음, 음성 대조군으로 정상 토끼 IgG를 넣고 37℃에서 1시간 반응시켰다. TBS-T로 세척하고, 염소 항-토끼 IgG 퍼옥시다제 접합체를 1/5000으로 희석하여 각 웰에 500㎕씩 분주한 다음, 37℃에서 1시간 반응시켰다. TBS-T로 세척하고, 기질-발색제(chromophore)를 50㎕씩 각 웰에 넣은 다음, 색이 변하면 약 10분 후 405nm에서 O.D.를 측정하였다. 다음 [표 3]은 그 결과를 수록한 것이다. After washing three times with TBS-T, IgM, IgG1, IgG2a, IgG2b, IgG3, IgA, Kappa, and Lambda were added to each well to be tested, and then normal rabbit IgG was added as a negative control and reacted at 37°C for 1 hour. After washing with TBS-T, goat anti-rabbit IgG peroxidase conjugate was diluted 1/5000 and 500㎕ was dispensed into each well, followed by reaction at 37°C for 1 hour. After washing with TBS-T, 50 ㎕ of substrate-chromophore was added to each well, and when the color changed, the O.D. was measured at 405 nm after about 10 minutes. The following [Table 3] contains the results.
상기 [표 3]에서 나타난 바와 같이, 상기 하이브리도마 3E1 세포주에서 생산된 단일클론 항체의 이소타입은 IgG2a/kappa이고, 상기 하이브리도마 4B9 세포주에서 생산된 단일클론 항체의 이소타입은 IgG1/kappa임을 확인하였다.As shown in [Table 3], the isotype of the monoclonal antibody produced in the hybridoma 3E1 cell line is IgG2a/kappa, and the isotype of the monoclonal antibody produced in the hybridoma 4B9 cell line is IgG1/kappa. It was confirmed that it was.
[ 실시예 3 ] 단일클론 항체의 생산 및 정제[Example 3] Production and purification of monoclonal antibodies
상기 하이브리도마 3E1 세포주와 4B9 세포주를 마우스의 복강 내로 주입하여 복수(ascites)가 생성되도록 유도하였다. 1주일 후에 각각의 하이브리도마 세포주를 마우스 1마리당 5x106씩 주사하고, 복강이 부풀어 오른 마우스에서 복수액을 채취하였다. 상기 복수액을 -20℃에서 얼린 다음, 얼지 않은 부분은 제거하였다. The hybridoma 3E1 cell line and the 4B9 cell line were injected into the abdominal cavity of mice to induce ascites. One week later, 5x10 6 of each hybridoma cell line was injected per mouse, and ascites fluid was collected from the mouse with a swollen abdominal cavity. The ascites fluid was frozen at -20°C, and then the unfrozen portion was removed.
상기 복수액을 다시 녹이고 암모늄설페이트[(NH4)2SO4]로 침전시킨 다음, 원심분리하여 침전물을 인산 완충액으로 용해하였다. 이어서 항체 정제용 결합 완충액(Pierce사, 미국)과 1:1로 섞어 준 후, protein A-agarose gel이 충전된 컬럼을 사용하여 정제하였다. 각 분획의 순도를 확인하기 위하여 SDS-PAGE를 수행하고, 그 결과를 첨부 도 3에 수록하였다. 50 kDa 부위와 25 kDa 부위에서 각각 항체의 중쇄(Heavy chain)와 경쇄(Light chain)가 관찰되어, 단일클론 항체의 분리 및 정제가 잘 이루어졌다고 판단하였다. The ascites fluid was re-dissolved and precipitated with ammonium sulfate [(NH 4 ) 2 SO 4 ], then centrifuged and the precipitate was dissolved in phosphate buffer. Next, it was mixed 1:1 with binding buffer for antibody purification (Pierce, USA) and purified using a column filled with protein A-agarose gel. SDS-PAGE was performed to confirm the purity of each fraction, and the results are shown in attached Figure 3. The heavy chain and light chain of the antibody were observed in the 50 kDa region and the 25 kDa region, respectively, and it was determined that the separation and purification of the monoclonal antibody was successful.
본 발명자들은 하이브리도마 3E1 세포주로부터 생산되는 단일클론 항체를 ‘3E1 항체’로 명명하고, 하이브리도마 4B9 세포주로부터 생산되는 단일클론 항체를 ‘4B9 항체’로 명명하였다. 상기 단일클론 항체 3E1은 10ml의 생산이 완료되었지만, 단일클론 항체 4B9는 생체 외 배양(in vitro culture)을 통해서 세포 배양액 100ml을 여과하여 항체를 확보하였다. 최종 생산된 각 항체의 농도와 양은 아래의 [표 4]와 같다.The present inventors named the monoclonal antibody produced from the hybridoma 3E1 cell line as ‘3E1 antibody’ and the monoclonal antibody produced from the hybridoma 4B9 cell line as ‘4B9 antibody’. Although 10 ml of monoclonal antibody 3E1 was produced, the monoclonal antibody 4B9 was obtained by filtering 100 ml of cell culture fluid through in vitro culture. The concentration and amount of each antibody finally produced are shown in [Table 4] below.
[ 시험예 1 ] 직접 ELISA를 이용한 항체의 민감도 시험[Test Example 1] Antibody sensitivity test using direct ELISA
항체 제조용 항원으로 사용한 재조합 KCa3.1 단백질을 검출용 단백질로 사용하여 최대 1ng/μl 부터 최소 0.0002ng/μl까지 농도별로 희석하고, 3E1 항체 및 4B9 항체의 민감도를 확인하였다. 다음 [표 5]는 그 결과를 나타낸 것으로, 대조군에 비해 본 발명에 따른 3E1 항체 및 4B9 항체의 반응성이 훨씬 우수하다는 것을 알수 있다. The recombinant KCa3.1 protein used as an antigen for antibody production was used as a detection protein and diluted by concentration from a maximum of 1ng/μl to a minimum of 0.0002ng/μl, and the sensitivity of the 3E1 antibody and 4B9 antibody was confirmed. The following [Table 5] shows the results, and it can be seen that the reactivity of the 3E1 antibody and 4B9 antibody according to the present invention is much better than that of the control group.
(μg/ml)(μg/ml)
[ 시험예 2 ] 샌드위치 ELISA에 따른 항원 결합활성 시험[Test Example 2] Antigen binding activity test according to sandwich ELISA
본 발명에 따른 4B9 항체를 포획항체(capture antibody)로 하고, 3E1 항체를 검출항체(detector antibody)로 사용하여 샌드위치 ELISA를 실시하였다. 구체적으로 96 웰 플레이트를 코팅 버퍼용액(Na2CO3, NaHCO3, pH 9.6) 내에 4μg/ml 농도 및 2μg/ml 농도의 4B9 항체로 하룻밤 동안 4℃에서 코팅하였다. 다음날 플레이트를 블로킹 용액으로 블로킹하였다. Sandwich ELISA was performed using the 4B9 antibody according to the present invention as a capture antibody and the 3E1 antibody as a detector antibody. Specifically, a 96-well plate was coated with 4B9 antibody at a concentration of 4 μg/ml and a concentration of 2 μg/ml in a coating buffer solution (Na 2 CO 3 , NaHCO 3 , pH 9.6) overnight at 4°C. The next day, the plate was blocked with blocking solution.
그 후 0.05% PBST, PBA(phosphate buffered saline내 0.1% BSA)로 희석된 다양한 농도의 KCa3.1 항원(10~0.15ng/μl)으로 2회 세척한 후, PBS(1:10,000 v/v) 내에 biotion을 접합한 3E1 항체를 각 웰에 첨가하였다. 세척 후 Avidin-HRP 100μl를 각 웰에 첨가하고 인큐베이션을 수행하였다. 최종적으로 TMB substrate reagent를 가하고 5분간 인큐베이션한 후 2.5 M H2SO4로 반응을 정지시켰다.Afterwards, wash twice with various concentrations of KCa3.1 antigen (10~0.15ng/μl) diluted with 0.05% PBST and PBA (0.1% BSA in phosphate buffered saline), followed by PBS (1:10,000 v/v). 3E1 antibody conjugated with biotion was added to each well. After washing, 100 μl of Avidin-HRP was added to each well and incubation was performed. Finally, TMB substrate reagent was added, incubated for 5 minutes, and the reaction was stopped with 2.5 MH 2 SO 4 .
그 결과, 다음 [표 6]과 같이, 3E1 항체는 높은 항원 결합활성을 나타냈으며, 0.05~10ng/ml의 KCa3.1까지 검출할 수 있었다. 즉, 본 발명의 단일클론 항체를 이용한 샌드위치 ELISA로 매우 미량의 KCa3.1 채널 단백질까지 검출할 수 있다는 것을 확인하였다. 이러한 결과는 본 발명의 3E1 항체 및 4B9 항체가 KCa3.1 채널 단백질의 발현 수준을 정밀하고 정확하게 검출할 수 있다는 것을 의미한다.As a result, as shown in [Table 6], the 3E1 antibody showed high antigen-binding activity and was able to detect up to 0.05 to 10 ng/ml of KCa3.1. In other words, it was confirmed that even very small amounts of KCa3.1 channel protein can be detected by sandwich ELISA using the monoclonal antibody of the present invention. These results mean that the 3E1 antibody and 4B9 antibody of the present invention can precisely and accurately detect the expression level of KCa3.1 channel protein.
서열목록 전자파일 첨부Sequence list electronic file attached
Claims (5)
A monoclonal antibody produced by the hybridoma 3E1 cell line with accession number KCTC 15261BP or the hybridoma 4B9 cell line with accession number KCTC 15262BP, and characterized by binding specifically to the human KCa3.1 channel protein.
Hybridoma 3E1 cell line, accession number KCTC 15261BP, which produces a monoclonal antibody that specifically binds to the human KCa3.1 channel protein.
Hybridoma 4B9 cell line, accession number KCTC 15262BP, which produces a monoclonal antibody that specifically binds to the human KCa3.1 channel protein.
B) 상기 재조합 벡터(pcDNA3.1-KCa3.1)를 발현시킨 ExpiCHO 세포주에서 재조합 KCa3.1 채널 단백질을 분리 및 정제하는 단계와;
C) 상기 재조합 KCa3.1 채널 단백질을 투입하여 면역을 유도한 마우스의 비장에서 B 림프구를 분리하고, 이를 골수종 세포와 혼합하여 하이브리도마를 제조하는 단계와;
D) 상기 하이브리도마 중에서 KCa3.1 채널 단백질에 특이적으로 반응하는 세포주를 클로닝하여 수탁번호 KCTC 15261BP인 3E1 세포주와 수탁번호 KCTC 15262BP인 4B9 세포주를 확보하는 단계와;
E) 상기 하이브리도마 3E1 세포주 또는 4B9 세포주를 배양하여 단일클론 항체를 수득하는 단계;
를 포함하는 것을 특징으로 하는, KCa3.1 채널 단백질에 특이적으로 결합하는 단일클론 항체의 제조방법.
A) preparing a recombinant vector (pcDNA3.1-KCa3.1) by inserting the gene for the KCa3.1 channel protein into the pcDNA3.1 expression vector;
B) isolating and purifying the recombinant KCa3.1 channel protein from the ExpiCHO cell line expressing the recombinant vector (pcDNA3.1-KCa3.1);
C) isolating B lymphocytes from the spleen of mice immunized with the recombinant KCa3.1 channel protein and mixing them with myeloma cells to prepare hybridomas;
D) cloning a cell line that specifically responds to the KCa3.1 channel protein among the hybridomas to obtain a 3E1 cell line with accession number KCTC 15261BP and a 4B9 cell line with accession number KCTC 15262BP;
E) culturing the hybridoma 3E1 cell line or 4B9 cell line to obtain a monoclonal antibody;
A method for producing a monoclonal antibody that specifically binds to the KCa3.1 channel protein, comprising:
Publications (1)
| Publication Number | Publication Date |
|---|---|
| KR20240110699A true KR20240110699A (en) | 2024-07-16 |
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