KR950008687A - Human leukocyte interferon α-2 recombinant expression vector and transformant - Google Patents

Human leukocyte interferon α-2 recombinant expression vector and transformant Download PDF

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KR950008687A
KR950008687A KR1019930019346A KR930019346A KR950008687A KR 950008687 A KR950008687 A KR 950008687A KR 1019930019346 A KR1019930019346 A KR 1019930019346A KR 930019346 A KR930019346 A KR 930019346A KR 950008687 A KR950008687 A KR 950008687A
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vkpm
chromatography
protein
cellulose
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KR960015746B1 (en
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지. 데바보브 블라디미르
알지라다스 브멜리스 블라다스
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블라다스 알지라디스 브멜리스
인스티튜트 오브 바이오테크놀로지 퍼멘터스
박완식
동신제약 주식회사
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
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    • C—CHEMISTRY; METALLURGY
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    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/78—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Pseudomonas

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Abstract

본 발명은 항바이러스성 인간 백혈구 인터페론 α-2 재조합 발현벡터, 그것으로 형질전환된 미생물 및 그로부터 생산된 인터페론 α-2를 정제하는 방법에 관한 것이다. 본 발명은 인간 백혈구 인터페론 α-2를 대량적으로 제조하기 위하여, 재조합 발현벡터로부터 형질전환된 생산균주를 탄소, 질소, 무기염류 및 생장 촉진제를 포함하는 중성 배지에서 항생제 존재하에 통기시키며 배양한 다음, 최종산물을 분리 정제하는 방법을 제공한다. 본 발명에서는 신규한 생산균주인 슈도모나스 균종 VG-86을 사용함으로써, 최종 산물인 인간 백혈구 인터페론 α-2의 수율을 높히고 간단한 분리과정을 통하여 효과적으로 분리, 정제할 수 있다.The present invention relates to an antiviral human leukocyte interferon α-2 recombinant expression vector, a microorganism transformed therewith and a method for purifying interferon α-2 produced therefrom. In order to prepare a large amount of human leukocyte interferon α-2, the production strain transformed from the recombinant expression vector is cultured with aeration in the presence of antibiotics in a neutral medium containing carbon, nitrogen, inorganic salts and growth promoters. It provides a method for separating and purifying the final product. In the present invention, by using the novel production strain Pseudomonas strain VG-86, it is possible to increase the yield of the final product human leukocyte interferon α-2 and to be effectively isolated and purified through a simple separation process.

Description

인간 백혈구 인터페론 α-2 재조합 발현벡터 및 형질전환체Human leukocyte interferon α-2 recombinant expression vector and transformant

본 내용은 요부공개 건이므로 전문내용을 수록하지 않았음Since this is an open matter, no full text was included.

제1도는 인터페론 α-2 유전자의 뉴클레오티드 서열을 나타낸다.1 shows the nucleotide sequence of the interferon a-2 gene.

제2도는 pVG3 프라스미드의 제한효소 지도를 나타낸다.2 shows a restriction map of pVG3 prasmid.

제3도는 본 발명의 pAYC105를 작제하는 과정을 나타내는 도식이다.3 is a diagram showing the process of constructing the pAYC105 of the present invention.

제4도는 면역친화성 크로마토그래피를 이용하여 인터페론 α-2를 정제하는 과정을 SDS-PAGE로 분석한 결과를 나타내는 사진이다.4 is a photograph showing the results of SDS-PAGE analysis of the purification of interferon α-2 using immunoaffinity chromatography.

Claims (10)

인간 알파 인터페론 α-2를 발현하는 플라스미드 벡터 pYAC105(VKPM B-6492).Plasmid vector pYAC105 (VKPM B-6492) expressing human alpha interferon a-2. 제1항의 pYAC105로 형질전환된 슈도모나스 퓨티다(Pseudomonas putida) VG-86(VKPM B-6492).Pseudomonas putida VG-86 (VKPM B-6492) transformed with pYAC105 of claim 1. (i) 슈도모나스 퓨티다(Pseudomonas putida) VG-86(VKPM B-6492)을 파괴하여 원심분리하는 공정; (ii) 전기 원심분리된 세포단백질추출물에 폴리에틸렌이민을 첨가하여 원심분리한 다음 얻은 상층액에 황산암모늄을 가하여 분획하는 공정; (iii) 전기 분획된 단백질을 페닐 실로크롬 S-80 크로마토그래피하는 공정 및 (iv) 전기 분획된 단백질을 단클론 항체를 가지는 흡착제 상에서 면역친화성 크로마토그래피하는 공정을 포함하는 인간 알파 인터페론 α-2의 제조방법.(i) destroying and centrifuging Pseudomonas putida VG-86 (VKPM B-6492); (ii) centrifugation by adding polyethylenimine to the cell centrifuged cell protein extract and then fractionating by adding ammonium sulfate to the obtained supernatant; (iii) chromatography of the electro-fractionated protein with phenyl silochrome S-80 and (iv) immunoaffinity chromatography of the electro-fractionated protein on an adsorbent with monoclonal antibodies. Manufacturing method. 제3항에 있어서, 면역친화성 크로마토그래피는 0.3M 염화나트륨을 포함하는 0..1M글리신(glycine) 완충용액(pH 3.0)을 이용하여 용출시키는 것을 특징으로 하는 방법.4. The method of claim 3, wherein the immunoaffinity chromatography is eluted with 0.1 M glycine buffer (pH 3.0) containing 0.3 M sodium chloride. (i) 슈도모나스 퓨티다(Pseudomonas putida) VG-86(VKPM B-6492)을 파괴하여 원심분리하는 공정; (ii) 전기 원심분리된 세포단백질추출물에 폴리에틸렌이민을 첨가하여 원심분리한 다음 얻은 상측액에 황산암모늄을 가하여 분획하는 공정; (iii) 전기 분획된 단백질을 페닐 실로크롬 S-80 크로마토그래피하고, ph 분획, 농축 및 투석하는 공정; (iv) 전기 분획된 단백질을 셀룰로오스 DE-52 이온 교환 크로마토그래피하는 공정; (v) 전기 분획된 단백질을 셀룰로오스 SM-52 이온 교환 크로마토그래피하는 공정; 및 (vi) 전기 분획된 단백질을 세파덱스 G-100 겔 여과 크로마토그래피하는 공정을 포함하는 인간 알파 인터페론 α-2의 제조방법.(i) destroying and centrifuging Pseudomonas putida VG-86 (VKPM B-6492); (ii) centrifugation by adding polyethyleneimine to the cell centrifuged cell protein extract and then fractionating by adding ammonium sulfate to the obtained supernatant; (iii) chromatographically fractionating the previously fractionated protein S-80, ph fractionating, concentrating and dialysis; (iv) cellulose DE-52 ion exchange chromatography of the electrically fractionated protein; (v) cellulose SM-52 ion exchange chromatography of the electrically fractionated protein; And (vi) Sephadex G-100 gel filtration chromatography of the electro-fractionated protein. 제5항에 있어서, 농축 및 투석은 10,000달톤의 투과한계를 가지는 PTGC형 카셋트를 이용하는 것을 특징으로 하는 방법.6. The method of claim 5, wherein the concentration and dialysis use a PTGC type cassette having a transmission limit of 10,000 Daltons. 제5항에 있어서, 셀룰로오스 DE-52 이온 교환 크로마토그래피는 0.04내지 0.06M 아세트산 완충용액(pH 5.0)을 이용하여 용출시키는 것을 특징으로 하는 방법.6. The method according to claim 5, wherein the cellulose DE-52 ion exchange chromatography is eluted using 0.04 to 0.06 M acetic acid buffer (pH 5.0). 제5항에 있어서, 셀룰로오스 SM-52 이온 교환 크로마토그래피는 0.1M 아세트산 완충용액과 0.3내지 0.5M 염화나트륨을 포함하는 0.1M 아세트산 완충용액(pH 4.8내지 5.2)를 이용하여 선형농도구배(lineargradient)로 용출시키는 것을 특징으로 하는 방법.6. The cellulose SM-52 ion exchange chromatography is performed in a linear gradient using 0.1M acetic acid buffer (pH 4.8 to 5.2) containing 0.1M acetic acid buffer and 0.3 to 0.5M sodium chloride. Eluting. 제3항의 방법에 따라 슈도모나스 퓨티다(Pseudomnas putida) VG-86(VKPM B-6492)으로 부터 제조된 인간 알파 인터페론 α-2.Human alpha interferon α-2 prepared from Pseudomnas putida VG-86 (VKPM B-6492) according to the method of claim 3. 제5항의 방법에 따라 슈도모나스 퓨티다(Pseudomnas putida) VG-86(VKPM B-6492)으로 부터 제조된 인간 알파 인터페론 α-2.Human alpha interferon α-2 prepared from Pseudomnas putida VG-86 (VKPM B-6492) according to the method of claim 5. ※ 참고사항 : 최초출원 내용에 의하여 공개하는 것임.※ Note: The disclosure is based on the initial application.
KR1019930019346A 1993-09-22 1993-09-22 RECOMBINANT VECTOR OF HUMAN LEUKOCYTE INTERFERON Ñß-2 KR960015746B1 (en)

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