KR20040072545A - Shuttle vector comprising a novel plasmid originated from Bifidobacterium longum MG1 - Google Patents

Shuttle vector comprising a novel plasmid originated from Bifidobacterium longum MG1 Download PDF

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KR20040072545A
KR20040072545A KR1020040057847A KR20040057847A KR20040072545A KR 20040072545 A KR20040072545 A KR 20040072545A KR 1020040057847 A KR1020040057847 A KR 1020040057847A KR 20040057847 A KR20040057847 A KR 20040057847A KR 20040072545 A KR20040072545 A KR 20040072545A
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지근억
박명수
강윤희
서정민
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주식회사 비피도
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Abstract

PURPOSE: A shuttle vector comprising a plasmid derived from Bifidobacterium longum MG1 is provided, thereby requiring no additional purification when a target gene is expressed by using the shuttle vector, and directly adding the expressed target gene into food or oral vaccines without further processing. CONSTITUTION: The shuttle vector comprises a plasmid pMG1 derived from Bifidobacterium longum MG1(KFCC-11273) having the nucleotide sequence set forth in SEQ ID NO:1 which is linked to a transformation vector derived from Escherichia coli selected from pEK104, pUC19 and pBR322, gene Mob derived from the plasmid pMG1 having the nucleotide sequence set forth in SEQ ID NO:2, gene Rep derived from the plasmid pMG1 having the nucleotide sequence set forth in SEQ ID NO:3, and a selection gene selected from ampicillin resistance gene, kanamycin resistance gene and chloramphenicol acetyl transferase gene.

Description

비피도박테리움 롱검 ＭＧ1 유래 신규 플라스미드를 함유하는 셔틀벡터{Shuttle vector comprising a novel plasmid originated from Bifidobacterium longum MG1}Shuttle vector comprising a novel plasmid originated from Bifidobacterium longum MG1}

본 발명은 비피도박테리움 롱검 MG1 유래 신규 플라스미드를 함유하는 셔틀벡터에 관한 것으로서, 보다 상세하게는 서열번호 1로 표시되는 염기서열을 갖는 플라스미드 pMG1과 대장균 유래 형질전환용 벡터를 연결하여 제조된, 대장균 및 비피도박테리움에서 상호 복제가 가능한 셔틀벡터; 상기 셔틀벡터에 목적 단백질을 코딩하는 목적 유전자를 삽입한 재조합 벡터; 및 상기 셔틀벡터 또는 상기 재조합 벡터에 비피도박테리움 유래 프로모터를 삽입한 재조합 벡터에 관한 것이다.The present invention relates to a shuttle vector containing a novel plasmid derived from Bifidobacterium longgum MG1, more specifically, prepared by linking a plasmid pMG1 having a nucleotide sequence represented by SEQ ID NO: 1 with a transforming vector derived from E. coli. Shuttle vectors capable of mutual replication in E. coli and Bifidobacterium; A recombinant vector inserting a target gene encoding a target protein into the shuttle vector; And a recombinant vector in which a Bifidobacterium-derived promoter is inserted into the shuttle vector or the recombinant vector.

인간의 장내에는 여러 종류의 미생물이 번식하면서 장내 세균총을 형성하고 있다. 이러한 장내 세균총의 조성과 활성은 인간 또는 동물의 건강뿐 만 아니라 영양, 생체 기능, 약물의 효율, 암화, 노화, 면역 반응, 감염에 대한 저항성 및 신체에 대한 다른 스트레스에도 광범위하게 영향을 미친다. 장내 세균총은 500개 이상의 서로 다른 세균종을 포함하며, 위 및 상부 소장에는 상대적으로 적은 수의 세균이 존재하고 있고 세균수는 대장으로 갈수록 증가한다. 상기 서술한 바와 같이, 장내 세균총은 인간의 건강에 포지티브(positive)한 영향을 주며, 장내 세균총 중 특히 비피도박테리움(Bifidobacterium)이 가장 중요한 것으로 알려져 있다. 이러한 사실은 비피도박테리움이 출생 이후로 일생 동안 사람의 대장 내에 서식하는 것을 관찰함으로써 알 수 있으며 또한, 우유를 먹이는 유아보다 모유를 먹이는 유아에서 비피도박테리움의 수가 더 많아 설사 질환에 걸릴 위험이 낮은 것으로 나타났다. 이러한 비피도박테리움의 수는 나이가 들수록 급격히 감소하는 것으로 나타났다.In the human intestine, various kinds of microorganisms multiply and form an intestinal flora. The composition and activity of these intestinal flora total not only affects the health of humans or animals, but also affects nutrition, biological function, drug efficiency, cancer, aging, immune response, resistance to infection and other stresses on the body. The intestinal flora contains over 500 different bacterial species, with a relatively small number of bacteria present in the stomach and upper small intestine, and the number of bacteria increases toward the large intestine. As described above, the intestinal bacterial flora has a positive effect on human health, and Bifidobacterium is known to be the most important among the intestinal bacterial flora. This can be seen by observing Bifidobacterium in the human large intestine after birth, and also because of the greater number of Bifidobacterium in breastfed infants than in milkfed infants. Found to be low. The number of Bifidobacterium decreased rapidly with age.

한편, 장내 균총의 분포상황은 연령, 인종, 생활환경, 음식성분 등 각종 요인에 의해 변화하고 있다. 특히, 장내 균총은 일반적으로 음식 종류에 의해 큰 영향을 받으며, 건강하게 오래 사는 장수자들 또는 성인병의 발병률이 낮은 사람들은 장내 유산균이 다른 사람에 비하여 많이 존재하는 것으로 보고되어 있다. 따라서, 정장 효과를 고양시키기 위한 음식의 중요성이 대두되고 있으며, 유산균 섭취를 통한 정장 효과를 얻기 위하여 최근에는 요구르트를 포함하는 종래의 유제품에 유산균을 첨가한 상품을 개발하는 연구가 활발히 진행되고 있다.On the other hand, the distribution of intestinal flora is changed by various factors such as age, race, living environment, and food ingredients. In particular, intestinal flora is largely influenced by food types, and long-lived healthy people or people with low incidence of adult diseases have been reported to have more intestinal lactic acid bacteria than other people. Therefore, the importance of food for enhancing the formal effect has emerged, and in order to obtain the formal effect through the ingestion of lactic acid bacteria, research has recently been actively conducted to develop products in which lactic acid bacteria are added to conventional dairy products including yogurt.

이와 같이 최근 건강에 대한 관심이 높아가면서 유산균을 건강식품 및 의약품 등에 사용하려는 많은 시도들이 이루어져 왔고, 실제로 다양한 유산균 식품 및 유산균 의약품이 개발되었다. 우리나라에서 사용되고 있는 유산균들은 모두 외국에서 개발된 것으로 우리나라 사람의 장내에 존재하는 균주들과 그 특성에 차이가 있을 수 있다. 또한, 우리나라 사람의 장내에만 존재하는 유산균은 지금까지 개발된 그 어느 유산균보다 우리나라 사람에게 더욱 적합하여 높은 생리 활성을 나타낼 수 있을 것으로 예상된다. 따라서, 우리나라 사람으로부터 유용한 한국형 유산균을 분리하여 우리나라 사람에게 적합한 새로운 유산균 식품 및 의약품 등을 개발하려는 시도가 이루어지게 되었다 .As the interest in health has recently increased, many attempts have been made to use lactic acid bacteria for health foods and medicines, and in fact, various lactic acid bacteria foods and lactic acid bacteria medicines have been developed. The lactic acid bacteria used in Korea are all developed in foreign countries, and there may be differences in the characteristics of the strains present in the intestines of Korean people. In addition, lactic acid bacteria present only in the intestines of Korean people is expected to be more suitable for Korean people than any other lactic acid bacteria developed so far can exhibit a high physiological activity. Therefore, an attempt has been made to separate useful Korean lactic acid bacteria from Korean people and develop new lactic acid bacteria foods and medicines suitable for Korean people.

비피도박테리움은 그람 양성균으로 Y 모양 또는 V 모양이며, 운동성이 없는 혐기성 균으로 포자를 형성하지 않는 발효균이다. 전통적으로 비피도박테리움은 젖산균에 속하는 것으로 분류되어 왔다. 비피도박테리움의 발효 주요 산물은 아세테이트 및 락테이트로 이들은 3:2의 비율로 생산되며, DNA의 GC 함유율은 55 내지 64%이다. 비피도박테리움은 숙주균에 특이적인 것으로 알려졌으며, 사람에서는 비피덤(B. bifidum), 롱검(B. longum), 브레베(B. breve), 인판티스(B. infantis) 및 아돌레센티스(B. adolescentis)가 발견되고 있으며, 상기 5 종류의 균주는 프로바이오틱스(probiotics)로 사용되고 있다. 프로바이오틱스란 인간이나 동물에 살아있는 미생물을 분말 또는 발효산물의 형태로 투여했을 때, 장내 미생물의 바람직한 균형 및 그들의 생리 특성을 증진시키며 숙주에게 유익한 효과를 나타내는 미생물 식품 강화제를 의미한다(Fuller, R.,Journal of Applied Biotechnology, 66:365-378, 1989).Bifidobacterium is a Gram-positive bacterium that is Y-shaped or V-shaped, and is a fermentation bacterium that does not form spores with anaerobic bacteria without motility. Traditionally, Bifidobacterium has been classified as belonging to lactic acid bacteria. The main products of fermentation of Bifidobacterium are acetate and lactate, which are produced in a ratio of 3: 2 and the GC content of DNA is 55 to 64%. Bifidobacterium have been known to be specific for the host bacterium, Bifidobacterium people in the bush (B. bifidum), ronggeom (B. longum), breve (B. breve), Infante Tees (B. infantis) and Adolfo Les sentiseu ( B. adolescentis ) has been found, and these five strains are used as probiotics. Probiotics are microbial food enhancers that, when administered to humans or animals, in the form of powders or fermented products, enhance the desired balance of intestinal microorganisms and their physiological properties and have a beneficial effect on the host (Fuller, R., Journal of Applied Biotechnology , 66: 365-378, 1989).

지금까지 젖산균인 락토바실러스(Lactobacillus) 및 락토코쿠스(Lactococcus)에서는 많은 벡터 시스템이 개발되었으며, 유전자 개량에 의한 상품화가 가까운 장래에 실현될 수 있을 것으로 전망되고 있다. 장내 정착성이 더욱 우수한 것으로 알려진 비피도박테리움의 벡터 시스템 개발은 아직 초보단계에 있다. 지금까지 비피도박테리움의 균종류에서 플라스미드에 대한 연구는 다음과 같다.Lactobacillus (lactic acid bacteria so far)Lactobacillus) And Lactococcus (Lactococcus), Many vector systems have been developed, and commercialization by genetic improvement is expected to be realized in the near future. The development of the vector system of Bifidobacterium, which is known to have better intestinal fixability, is still in its infancy. until now Studies on plasmids in Bifidobacterium spp. Are as follows.

먼저, 처음에는 사람에서 분리된 균 중에서 롱검(B. longum)에만 플라스미드가 존재한다고 보고되었고, 상기 균주로부터 분리한 플라스미드를 대장균 유래 형질전환용 벡터와 연결한 셔틀벡터가 개발되었다. 또한, 글로보숨(B. globosum),아스테로이드(B. asteroides),인디컴(B. indicum) 등에도 플라스미드가 존재하는 것으로 보고되었으며, 상기 균주들은 전기영동 패턴 또는 서던 혼성화(Southern hybridization) 등을 통하여 상동성에 따라 그룹으로 나누어 졌다. 또 다른 균주인 브레베(B. breve)에도 플라스미드가 존재한다고 보고된 바 있다(Sgorbati, B.et al,Microbiologica., 6:169-173, 1983; Tannock, G. W.et al.,Microbiology, 28:1225-1228, 1990).First, among the bacteria isolated from humans, long gum (B. longumOnly plasmids were reported to be present, and shuttle vectors linking plasmids isolated from the strains to E. coli-derived transformation vectors were developed. In addition, Globosum (B. globosum),Asteroid (B. asteroides),IndicomB. indicum) It has been reported that the plasmid is also present in the back, the strains were divided into groups according to homology through the electrophoresis pattern or Southern hybridization. Another strain, breve (B. breveHas also been reported to have a plasmid (Sgorbati, B.).et al,Microbiologica, 6: 169-173, 1983; Tannock, G. W.et al.,Microbiology, 28: 1225-1228, 1990).

비피도박테리움의 플라스미드를 이용한 벡터 개발은 1990년대에 들어서야 보고되기 시작하였다. 롱검(B. longum)B2577의 1.9kb 플라스미드 pMB1을 대장균 벡터에 클로닝하여 제한효소지도가 작성되었다. 1994년에는 pMB1을 기본 벡터로 하여 대장균과 롱검(B. longum)에서 상호 복제되는 셔틀벡터인 pRM2가 개발되었으며, 전기천공법(electroporation)에 의해 롱검(B. longum)을 형질전환(transformation)하는데 성공하였다. 또한, 최근에는 상기 pMB1의 전체 염기서열이 밝혀졌다(Mateuzzi, D.,et al.,Letters in Applied Microbiology, 11:220-223, 1990; Missichi, R.,et al,Plasmid, 32:208-211, 1994; Argnami, A.et al.,Microbiology, 142:109-114, 1996). 비피도박테리움은 두꺼운 다층의 세포벽과 여러 종류의 펩티도글라이칸(peptidoglycan), 다당류(polysaccharide), 리포테이코산(lipoteichoic acid) 및 단백질로 구성된 세포벽을 갖고 있으며, 이러한 세포벽의 특성은 외부 DNA 분자를 받아들이는데 장벽이 되어왔다. 비피도박테리움을 형질전환 시키기 위하여 학문적 또는 산업적인 차원에서 다양한 시도가 이루어졌으나 성공된 바가 드물었으며, 롱검(B. longum)을 전기천공법을 이용하여 낮은 효율로나마 형질전환체를 제조한 연구가 보고된 바 있다(Missich, R.et al.,Plasmid, 32:208-211, 1994).Vector development using Bifidobacterium plasmids was not reported until the 1990s. A restriction enzyme map was generated by cloning the 1.9 kb plasmid pMB1 of B. longum B2577 into an E. coli vector. In 1994, pRM2, a shuttle vector cloned from Escherichia coli and B. longum , was developed using pMB1 as a base vector, and transformed into B. longum by electroporation. Succeeded. In addition, the entire nucleotide sequence of the pMB1 has recently been revealed (Mateuzzi, D., et al ., Letters in Applied Microbiology , 11: 220-223, 1990; Missichi, R., et al , Plasmid , 32: 208- 211, 1994; Argnami, A. et al ., Microbiology , 142: 109-114, 1996). Bifidobacterium has a thick, multi-layered cell wall and a cell wall composed of several types of peptidoglycans, polysaccharides, lipoteichoic acid, and proteins, which characterize external DNA molecules. It has been a barrier to accepting. Various attempts have been made at the academic or industrial level to transform Bifidobacterium, but few have been successful. A researcher producing a transformant with low efficiency using electroporation using long gum ( B. longum ) Have been reported (Missich, R. et al ., Plasmid , 32: 208-211, 1994).

상기에서 분리된 플라스미드 pMB1을 이용하여 셔틀벡터 pNC7을 제조하였으며, 다양한 종의 비피도박테리움에 이용할 수 있는 형질전환 방법을 개발하였고, 그 효율은 균에 따라서 1.0 ×101에서부터 1.2 × 105CFU/㎍ DNA 까지 다양하게 나타났다. 상기 형질전환 방법에 있어서 악틸라이트 P(ActilightⓡP)를 배지의 당원으로 사용하였고, 대수 증식기 초기에 균을 회수하여 컴피턴트 세포(competent cell)를 제조하였으며, 전기천공법의 조건을 12.5 kV/cm, 100 ℃, 25 ㎌으로 조정하여 형질전환을 수행하였다. 일본에서는 비피도박테리움 롱검(B. longum)에서 분리된 3.6kb 플라스미드(pTB6,표 1참고)를 이용하여 대장균과 비피도박테리움에서 상호 복제되는 셔틀벡터를 개발한 바 있다(Matsummura, H.et al.,Biosci. biotech. Biochem., 61(7):1211-1212, 1997).The shuttle vector pNC7 was prepared using the isolated plasmid pMB1, and a transformation method that can be used for Bifidobacterium of various species was developed, and the efficiency thereof was 1.0 × 10 depending on the bacteria.OneFrom 1.2 × 105CFU / μg DNA Appeared to vary. Actilite P (Actilight in the transformation methodⓇP) was used as the glycoside of the medium, and the cells were recovered at the beginning of the logarithmic growth period to prepare competent cells. Was performed. In Japan, Bifidobacterium longgum (B. longum)3.6kb plasmid isolated from (pTB6,Table 1Has developed a shuttle vector that replicates in Escherichia coli and Bifidobacterium (Matsummura, H.).et al.,Biosci. biotech. Biochem, 61 (7): 1211-1212, 1997).

우리나라에서는 1994년에 처음으로 비피도박테리움에서 플라스미드를 분리하였고, 서던 혼성화(Southern hybridization)에 의하여 몇 가지 상동 그룹으로 분류하였다(정현서 등, 한국식품과학회 춘계 학술 발표, 1994; 이주훈 등, 한국식품과학회 춘계 학술 발표, 1997). 이들 중 두 균주는 각각 에리트로마이신(erythromycin) 및 테트라싸이클린(tetracyclin)에 대하여 상대적으로 강한 내성을 보였으며, 플라스미드가 소실되면 내성도 현저히 감소하는 것을 확인하였다. 또 다른 균주인 비피도박테리움 롱검(B. longum)KJ는 두 개의 서로 다른 플라스미드인 pKJ36 및 pKJ50(표 1참고)을 보유하고 있었다. 이들을 대장균 유래 형질전환용 벡터에 클로닝하여 이를 바탕으로 두 개의 플라스미드에 대한 유전자 지도를 작성하였으며, 전체 염기서열을 밝히고 이를 비교 분석하였다(박명수 등, 한국 산업미생물학회 추계 학술 발표, 1995, 1996, 1997). 그 결과 두 개의 플라스미드는 매우 유사한 구조적 특징이 있었고, 그램 양성 및 그램 음성 미생물들의 플라스미드에서 발현되는 Rep 단백질 및 Mob 단백질과 아미노산 상동성을 가지는 ORF를 포함하고 있음을 알 수 있었다. 상기 각각의 ORF는 번역/전사(translation/transcription) 수준에서 발현되는 것이 확인되었다. 이와 같은 분석자료를 바탕으로 대장균과 비피도박테리움에서 상호 복제되는 셔틀벡터 pBKJ50F, pBKJ50R 및 pBRepA를 제조하여 비피도박테리움을 형질전환하는 데 사용하였다.In Korea, plasmids were isolated from Bifidobacterium for the first time in 1994 and classified into several homologous groups by Southern hybridization (Jun Hyun Seo, et al., Spring Conference, Korean Food Science Association, 1994; Spring Institute of Science, 1997). Two of these strains showed relatively strong resistance to erythromycin and tetratracyclin, respectively, and it was confirmed that the resistance was also significantly reduced when the plasmid was lost. Another strain, Bifidobacterium longgum ( B. longum) KJ, had two different plasmids, pKJ36 and pKJ50 (see Table 1 ). These were cloned into E. coli-derived transformation vectors, and genetic maps of the two plasmids were generated, and the entire sequencing was identified and compared. ). As a result, the two plasmids had very similar structural features and contained ORFs having amino acid homology with Rep protein and Mob protein expressed in plasmids of gram positive and gram negative microorganisms. Each ORF was found to be expressed at the translation / transcription level. Based on the analytical data, shuttle vectors pBKJ50F, pBKJ50R, and pBRepA were cloned from E. coli and Bifidobacterium, and used to transform Bifidobacterium.

상기에서 살펴본 비피도박테리움의 플라스미드를 이용하여 개발된 벡터들은 모두가 외래의 항생제 내성 유전자를 선별 유전자(selection marker)로 함유하고 있어 이들을 직접 식품에 사용할 수 없는 단점이 있다. 따라서, 식품에 안전하게 사용할 수 있는 식품 등급용 벡터(food-grade vector)의 개발이 요구되고 있다. 이를 위해 항생제 내성 유전자들을 제거하고 식품 등급(food grade)으로 사용할 수 있는 선별 유전자로 대체할 뿐만 아니라 외래 유전자의 발현을 조절할 수 있는 프로모터/오퍼레이터(promoter/operator)등을 삽입함으로써 새로운 셔틀벡터의 개발이 진행 중에 있다. 하기표 1에 비피도박테리움에 포함된 플라스미드 및 이로부터 개발된 셔틀벡터를 나타내었다.The vectors developed using the Bifidobacterium plasmid described above all have a disadvantage that they cannot be directly used in food because they contain foreign antibiotic resistance genes as selection markers. Therefore, there is a need for developing a food-grade vector that can be safely used in food. To this end, the development of a new shuttle vector by removing antibiotic resistance genes and replacing them with a selection gene that can be used as a food grade, as well as by inserting a promoter / operator that can regulate the expression of foreign genes. This is in progress. Table 1 below shows the plasmids contained in Bifidobacterium and the shuttle vectors developed therefrom.

플라스미드(kb)Plasmid (kb) 숙주 세포Host cell 벡터vector 선별 유전자Screening genes pMB1(1.9)pMB1 (1.9) 비피도박테리움 롱검Bifidobacterium longgum pRM2pRM2 스펙티노마이신(spectinomycin)a암피실린(ampicillin)b Spectinomycin a ampicillin b pNC7pNC7 클로람페니콜(chloramphenicol)a암피실린b Chloramphenicol a ampicillin b pKJ36(3.6)pKJ36 (3.6) 비피도박테리움 롱검 KJBifidobacterium longgum KJ pEKJ36pEKJ36 클로람페니콜a암피실린b Chloramphenicol a Ampicillin b pKJ50(5.0)pKJ50 (5.0) 비피도박테리움 롱검 KJBifidobacterium longgum KJ pBKJ50FpBKJ50F 클로람페니콜a,b Chloramphenicol a, b pBKJ50RpBKJ50R 클로람페니콜a,b Chloramphenicol a, b pBRepApBRepA pNBb1(5.6)pNBb1 (5.6) 비피도박테리움 브레베Bifidobacterium breve 개발되지 않았음Not developed pTB6(3.6)pTB6 (3.6) 비피도박테리움 롱검Bifidobacterium longgum pBLES100pBLES100 스트랩토마이신(streptomycin)a암피실린b Streptomycin a Ampicillin b

a: 비피도박테리움에 대한 선별 유전자 a: selection gene for Bifidobacterium

b: 대장균에 대한 선별 유전자b: selection gene for E. coli

이에 본 발명자들은 상기와 같은 문제점들을 해결하면서 목적 유전자를 비피도박테리움에서 발현시 별도의 정제 과정 없이도 식품에 첨가할 수 있는 식품 첨가물 또는 경구용 백신의 제조에 사용될 수 있는, 대장균 및 비피도박테리움에서 상호 복제가 가능하면서 목적 유전자를 발현시킬 수 있는 셔틀벡터 및 상기 목적 유전자를 비피도박테리움 내에서 강하게 발현시킬 수 있는 프로모터를 개발함으로써본 발명을 완성하게 되었다.Therefore, the present inventors solve the problems as described above, E. coli and Bifidobacte can be used in the manufacture of food additives or oral vaccines that can be added to food without a separate purification process when the target gene is expressed in Bifidobacterium The present invention was completed by developing a shuttle vector capable of expressing a gene of interest and a promoter capable of strongly expressing the gene of interest in Bifidobacterium.

따라서, 본 발명의 목적은 대장균 및 비피도박테리움에서 상호 복제가 가능한 셔틀벡터를 제공하는 것이다.Therefore, it is an object of the present invention to provide a shuttle vector capable of mutual replication in Escherichia coli and Bifidobacterium.

본 발명의 다른 목적은 상기 셔틀벡터에 목적 단백질을 코딩하는 목적 유전자를 삽입한 재조합 벡터 및 상기 재조합 벡터로 형질전환된 형질전환체를 제공하는 것이다.It is another object of the present invention to provide a recombinant vector in which a target gene encoding a target protein is inserted into the shuttle vector, and a transformant transformed with the recombinant vector.

본 발명의 또 다른 목적은 상기 셔틀벡터 또는 상기 재조합 벡터에 비피도박테리움 내에서 목적 유전자를 고효율로 발현시킬 수 있는 비피도박테리움에서 분리한 프로모터를 삽입한 재조합 벡터 및 상기 재조합 벡터로 형질전환된 형질전환체를 제공하는 것이다.Still another object of the present invention is to transform the shuttle vector or the recombinant vector into a recombinant vector and a recombinant vector in which a promoter isolated from Bifidobacterium capable of expressing a target gene in Bifidobacterium with high efficiency is inserted. It is to provide a transformant.

도 1은 비피도박테리움 롱검 MG1(Bifidobaterium longumMG1)에서 분리한 pMG1의 개략적인 개열지도를 나타낸 것이고, Figure 1 shows a schematic cleavage map of pMG1 separated from Bifidobaterium longum MG1 ( Bifidobaterium longum MG1 ) ,

도 2는 대장균과 비피도박테리움 내에서 상호 복제가 가능한 셔틀벡터 pBES2의 제조 과정을 나타낸 것이며, Figure 2 shows the manufacturing process of the shuttle vector pBES2 that can be mutually cloned in E. coli and Bifidobacterium,

도 3은 도 2에서 제조된 셔틀벡터 pBES2를 이용하여 목적 유전자로 아밀라아제 유전자를 삽입한 재조합 벡터 pYBamy59의 제조 과정을 나타낸 것이고, FIG. 3 illustrates a process for preparing a recombinant vector pYBamy59 in which an amylase gene is inserted into a target gene using the shuttle vector pBES2 prepared in FIG. 2.

도 4는서열번호 4또는서열번호 5로 표시되는 염기서열을 갖는 비피도박테리움 GE65(Bifidobaterium sp. GE65)의 프로모터를 포함하는 pbifGPF7 또는 pbifGPF10를 분리하는 과정을 나타낸 것이며, 4 illustrates a process of separating pbifGPF7 or pbifGPF10 comprising a promoter of Bifidobaterium sp . GE65 having a nucleotide sequence represented by SEQ ID NO: 4 or SEQ ID NO: 5 ,

도 5는 도 4에서 제조한 pbifGPF7 또는 pbifGPF10에서 프로모터 및 GFP를 분리하여 pBES2에 삽입한 pYBGFP7 또는 pYBGFP10의 제조 과정을 나타낸 것이고, FIG. 5 illustrates a process of preparing pYBGFP7 or pYBGFP10 separated from a promoter and GFP in pbifGPF7 or pbifGPF10 prepared in FIG. 4 and inserted into pBES2.

도 6은 셔틀벡터 pBES2의 제조에 이용되는 pEK104 벡터의 개략적인 개열지도를 나타낸 것이다. 6 shows a schematic cleavage map of the pEK104 vector used in the preparation of the shuttle vector pBES2.

상기와 같은 본 발명의 목적을 달성하기 위하여, 본 발명은서열번호 1로 표시되는 염기서열을 갖는 플라스미드 pMG1과 대장균 유래 형질전환용 벡터를 연결하여 제조된, 대장균 및 비피도박테리움에서 상호 복제가 가능한 셔틀벡터를 제공한다.In order to achieve the object of the present invention as described above, the present invention is produced by connecting the plasmid pMG1 having the nucleotide sequence represented by SEQ ID NO: 1 and E. coli-derived transformation vector, mutual replication in E. coli and Bifidobacterium Provides possible shuttle vectors.

본 발명의 다른 목적을 달성하기 위하여, 본 발명은 상기 셔틀벡터에 목적 단백질을 코딩하는 목적 유전자를 삽입한 재조합 벡터 및 상기 재조합 벡터로 형질전환된 비피도박테리움 속 미생물을 제공한다.In order to achieve another object of the present invention, the present invention provides a recombinant vector inserting the target gene encoding the target protein in the shuttle vector and the Bifidobacterium genus microorganism transformed with the recombinant vector.

또한 본 발명의 또 다른 목적을 달성하기 위하여, 본 발명은 상기 셔틀벡터 또는 상기 재조합 벡터에 서열번호 4 또는 서열번호 5로 표시되는 염기서열을 갖는 비피도박테리움 GE65(Bifidobacterumsp. GE65)의 프로모터를 삽입한 재조합 벡터 및 상기 재조합 벡터로 형질전환된 비피도박테리움 속 미생물을 제공한다.In addition, to achieve another object of the present invention, the present invention is a promoter of Bifidobacterium GE65 ( Bifidobacterum sp. GE65) having a nucleotide sequence represented by SEQ ID NO: 4 or SEQ ID NO: 5 in the shuttle vector or the recombinant vector It provides a recombinant vector inserted into the Bifidobacterium microorganism transformed with the recombinant vector.

이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.

종래 형질전환 숙주세포로 주로 사용되는 대장균에서 목적 단백질을 발현시켜 이를 식품 첨가물 또는 경구용 백신으로 사용하기 위해서는, 대장균 단백질의 독성 등을 제거하기 위한 별도의 정제과정이 필요하기 때문에, 사람에 독성을 나타내지 않는 비피도박테리움을 숙주세포로 사용하려는 연구가 진행되어 왔다. 또한, 비피도박테리움의 플라스미드를 이용하여 개발된 벡터들은 모두 외래의 항생제 내성 유전자를 선별 유전자(selection marker)로 함유하고 있어서 이들을 직접 식품에 사용할 수 없었다. 본 발명은 이러한 문제점을 해결하여 식품에 안전하게 사용할 수 있는 식품 등급용 벡터(food-grade vector)를 제공하는데 특징이 있다.To express the target protein in Escherichia coli, which is mainly used as a transgenic host cell, and to use it as a food additive or oral vaccine, a separate purification process is required to remove the toxicity of E. coli protein. Research has been conducted to use Bifidobacterium, which is not shown, as a host cell. In addition, all of the vectors developed using Bifidobacterium plasmids contained foreign antibiotic resistance genes as selection markers, so they could not be directly used in food. The present invention is characterized by providing a food-grade vector that can be used safely in food by solving these problems.

따라서, 본 발명은서열번호 1로 표시되는 염기서열을 갖는 플라스미드 pMG1과 대장균 유래 형질전환용 벡터를 연결하여 제조된, 대장균 및 비피도박테리움에서 상호 복제가 가능한 셔틀벡터를 제공한다.Therefore, the present invention provides a shuttle vector capable of mutual replication in Escherichia coli and Bifidobacterium, which is prepared by linking a plasmid pMG1 having a nucleotide sequence represented by SEQ ID NO: 1 with an E. coli-derived transformation vector.

본 발명의 셔틀벡터에 있어서, 상기 플라스미드 pMG1은 비피도박테리움 롱검 MG1(Bifidobacterium longumMG1(기탁번호 KFCC-11273)에서 유래된 것을 특징으로한다. 본 발명의 셔틀벡터는 플라스미드 pMG1에서 유래된서열번호 2로 표시되는 염기서열을 갖는Mob유전자, 플라스미드 pMG1에서 유래된서열번호 3으로 표시되는 염기서열을 갖는Rep유전자 및 선별 유전자를 포함한다. 또한 본 발명의 셔틀벡터에 있어서, 상기 대장균 유래 형질전환용 벡터는 pEK104(고려대학교 생명공학원 유전생화학실험실, 장효일 교수에게서 분양 받음), pUC19(Clontech) 및 pBR322(Clontech)로 이루어진 군에서 선택되는 어느 하나인 것이 바람직하다. 구체적으로, 본 발명에서는 플라스미드 pMG1과 pEK104를 연결하여 제조된 셔틀벡터 pBES2를 제공한다. 상기 셔틀벡터 pBES2는서열번호 2로 표시되는Mob유전자,서열번호 3으로 표시되는Rep유전자 및 선별 유전자를 포함한다. 셔틀벡터 pBES2의 개략적인 개열지도와 제조과정이도 2에 도시되어 있다. 본 발명에 따른 셔틀벡터 pBES2는 30회 정도의 세포분열 후에도 세포 안에서 유지되는 높은 안정성을 보였다.In the shuttle vector of the present invention, the plasmid pMG1 is derived from Bifidobacterium longum MG1 (Accession No. KFCC-11273). The shuttle vector of the present invention is a sequence number derived from plasmid pMG1. Mob gene having a nucleotide sequence represented by 2 , Rep gene having a nucleotide sequence represented by SEQ ID NO: 3 derived from the plasmid pMG1, and a selection gene is included in the shuttle vector of the present invention, E. coli-derived transformation for The vector is preferably any one selected from the group consisting of pEK104 (Genetic Biochemistry Laboratory, Korea University, Professor Hyo Il Chang), pUC19 (Clontech) and pBR322 (Clontech). To provide a shuttle vector pBES2 prepared by connecting pEK104. The shuttle vector pBES2 is Mo represented by SEQ ID NO: 2 . b gene, Rep gene represented by SEQ ID NO: 3 , and a selection gene, a schematic cleavage map and a manufacturing process of the shuttle vector pBES2 is shown in Figure 2. Shuttle vector pBES2 according to the present invention is about 30 times a cell High stability was maintained in cells even after division.

본 발명의 셔틀벡터에 포함되는 선별 유전자로는 암피실린(ampicillin) 저항성 유전자, 가나마이신(kanamycin) 저항성 유전자 및 클로람페니콜 아세틸 트랜스퍼라아제(chloramphenicol acetyl transferase) 유전자로 이루어진 군에서 선택되는 하나 이상인 것이 바람직하다. 구체적으로, 본 발명에서는 암피실린 저항성 유전자 및 클로람페니콜 아세틸 트랜스퍼라아제 유전자를 사용하였다.The selection gene included in the shuttle vector of the present invention is preferably at least one selected from the group consisting of ampicillin resistance gene, kanamycin resistance gene, and chloramphenicol acetyl transferase gene. Specifically, the ampicillin resistance gene and chloramphenicol acetyl transferase gene were used in the present invention.

또한 본 발명은 본 발명의 셔틀벡터에 목적 단백질을 코딩하는 목적 유전자를 삽입한 재조합 벡터를 제공한다.The present invention also provides a recombinant vector in which the target gene encoding the target protein is inserted into the shuttle vector of the present invention.

상기 목적 유전자는 아밀라아제 유전자, 백신유전자, 항암유전자 및 다양한 생리활성을 나타낼 수 있는 유전자로 이루어진 군으로부터 선택되는 어느 하나인 것이 바람직하다. 구체적으로, 본 발명에서는 셔틀벡터 pBES2에 목적 유전자로 아밀라아제 유전자를 삽입한 재조합 벡터 pYBamy59를 제조하였다. 재조합 벡터 pYBamy59는 크기가 10.1 kb이고, 그 개략적인 개열지도와 제조과정이도 3에 도시되어 있다.The target gene is preferably any one selected from the group consisting of amylase gene, vaccine gene, anticancer gene and genes capable of exhibiting various physiological activities. Specifically, in the present invention, a recombinant vector pYBamy59 was prepared in which the amylase gene was inserted into the shuttle vector pBES2. PYBamy59 recombinant vector is a size 10.1 kb, is shown in the schematic cleavage map in Fig. 3 the manufacturing process.

또한 본 발명자들은 셔틀벡터의 제조에 사용하였던 플라스미드를 분리한 비피도박테리움을 목적 유전자가 삽입된 재조합 벡터로 형질전환함으로써 형질전환체가 목적 유전자를 높은 효율로 발현시킬 수 있음을 제시하였다. 따라서, 본 발명은 상기 재조합 벡터로 형질전환된 비피도박테리움 속 미생물을 제공한다. 형질전환 방법을 구체적으로 설명하면 다음과 같다.In addition, the present inventors have suggested that the transformant can express the target gene with high efficiency by transforming the Bifidobacterium from which the plasmid used for the preparation of the shuttle vector is isolated with the recombinant vector into which the target gene is inserted. Accordingly, the present invention provides a Bifidobacterium genus microorganism transformed with the recombinant vector. The transformation method is described in detail as follows.

먼저, 비피도박테리움 유래 플라스미드와 대장균 유래 형질전환용 벡터를 연결하여 비피도박테리움과 대장균에서 상호 복제되는 셔틀벡터를 제조한다. 그리고 나서, 상기 셔틀벡터에 목적 단백질을 코딩하는 목적 유전자를 결합시켜 재조합 벡터를 제조한다. 재조합 벡터를 제조하는 방법으로는 본 기술분야에서 일반적으로 알려진 공지의 방법이 사용될 수 있다. 이후, 상기 재조합 벡터로 형질전환시키기 위한 숙주세포로서 상기 셔틀벡터의 제조에 이용된 비피도박테리움을 이용한다는 데 본 방법의 특징이 있다. 이렇게 재조합 벡터로 형질전환시키기 위한 숙주세포로 재조합 벡터의 제조에 이용된 셔틀벡터가 유래된 원래의 비피도박테리움을 이용함으로써 형질전환 효율을 크게 향상시킬 수 있었다. 구체적으로, 본 발명에서는 재조합 벡터의 제조에 이용된 셔틀벡터가 유래된 비피도박테리움으로 컴피턴트 세포를 제조함과 동시에, 형질전환시 옥시레이즈(oxyrase) 효소를 처리함으로써 형질전환 효율을 10배 이상 증가시킬 수 있었다.First, by connecting the Bifidobacterium-derived plasmid and E. coli-derived transformation vector to prepare a shuttle vector that is replicated in the Bifidobacterium and E. coli. Then, the recombinant vector is prepared by binding the shuttle vector to the target gene encoding the target protein. As a method for preparing a recombinant vector, known methods generally known in the art may be used. Thereafter, the present invention is characterized by using the Bifidobacterium used in the preparation of the shuttle vector as a host cell for transformation with the recombinant vector. As a host cell for transforming into a recombinant vector, the transformation efficiency was greatly improved by using the original Bifidobacterium derived from the shuttle vector used in the preparation of the recombinant vector. Specifically, the present invention prepares competent cells with the Bifidobacterium derived shuttle vector used in the preparation of the recombinant vector, and at the same time by transforming the oxyrase enzyme at the time of transformation by 10 times transformation efficiency Could increase over.

본 발명에 따른 재조합 벡터 pYBamy59로 형질전환된 비피도박테리움 롱검 MG1은 2001년 2월 24일자로 한국종균협회에 기탁번호 KFCC-11274로 기탁되었다.The Bifidobacterium longgum MG1 transformed with the recombinant vector pYBamy59 according to the present invention was deposited on February 24, 2001, with the accession number KFCC-11274 to the Korean spawn association.

나아가, 본 발명은 본 발명의 셔틀벡터 또는 상기 셔틀벡터에 목적 유전자를 삽입한 재조합 벡터에서열번호 4또는서열번호 5로 표시되는 염기서열을 갖는 비피도박테리움 GE65(Bifidobaterium sp. GE65)의 프로모터를 삽입하여 제조한 재조합 벡터 및 상기 재조합 벡터로 형질전환된 비피도박테리움 속 미생물을 제공한다. 본 발명자들은 비피도박테리움 내에서 목적 유전자의 발현 효율을 더욱 증가시키기 위하여 비피도박테리움 GE65로부터 활성이 강한 프로모터(promoter)를 분리하게 되었다. 상기 활성이 강한 프로모터를 분리할 수 있는 균주로는 비피도박테리움 GE65, 비피도박테리움 롱검 MG1, 비피도박테리움 아돌레센티스 INT57 또는 비피도박테리움 롱검 ATCC15707을 사용하는 것이 바람직하며, 본 발명에서는 비피도박테리움 GE65를 사용하여서열번호 4또는서열번호 5로 표시되는 프로모터를 분리하였다. 비피도박테리움의 프로모터를 스크리닝하는 방법으로는 당업계에서 알려진 통상의 방법이 이용될 수 있으나, 구체적으로, 본 발명에서는 리포터 유전자로 사용되고 있는 gfp 유전자를 이용하여 스크리닝하였다.Furthermore, the present invention provides a promoter of Bifidobacterium GE65 having a nucleotide sequence represented by SEQ ID NO: 4 or SEQ ID NO: 5 in a shuttle vector of the present invention or a recombinant vector into which the target gene is inserted into the shuttle vector. It provides a recombinant vector prepared by inserting the Bifidobacterium microorganism transformed with the recombinant vector. The present inventors have isolated a highly active promoter from Bifidobacterium GE65 in order to further increase the expression efficiency of the gene of interest in Bifidobacterium. As a strain capable of separating the active promoter, it is preferable to use Bifidobacterium GE65, Bifidobacterium longgum MG1, Bifidobacterium adolescents INT57 or Bifidobacterium longgum ATCC15707. In Bifidobacterium GE65 was used to isolate a promoter represented by SEQ ID NO: 4 or SEQ ID NO: 5 . As a method for screening a promoter of Bifidobacterium, conventional methods known in the art may be used. Specifically, in the present invention, screening is performed using a gfp gene which is used as a reporter gene.

프로모터와 결합하는 프로모터 탐침 벡터(promoter probe vector)를 제조한 후, 상기 프로모터 탐침 벡터를 이용하여 대장균 DH5α에서 GFP를 발현하는 프로모터가 삽입되어 있는 벡터 pbifGFP1, 2, 4, 5, 6, 7 및 10을 각각 분리하였고, 이들 중 프로모터 활성이 상대적으로 강한서열번호 4로 표시되는 PG7 프로모터를 포함하는 벡터 pbifGFP7 및서열번호 5로 표시되는 PG10을 포함하는 벡터 pbifGFP10을 얻었다.도 4에 프로모터 탐침 벡터의 개략적인 제조과정과 벡터 pbifGFP7 또는 pbifGFP10의 개략적인 개열지도가 도시되어 있다. 상기 벡터 pbifGFP7 또는 pbifGFP10을 포함하는 대장균의 GFP 발현은 UV 파장을 조사하여 녹색 형광을 확인함으로써 선별할 수 있다. 구체적으로, 상기 벡터 pbifGFP7 또는 pbifGFP10과 본 발명에 따른 셔틀벡터 pBES2를 재조합하여 본 발명에 따른 프로모터 PG7 또는 PG10으로 목적 유전자를 발현시킬 수 있는 벡터 pYBGFP7 또는 pYBGFP10을 제조하였다. 상기 벡터 pYBGFP7 또는 pYBGFP10의 개략적인 개열지도가도 5에 도시되어 있다.After preparing a promoter probe vector that binds to a promoter, the vectors pbifGFP1, 2, 4, 5, 6, 7, and 10 into which a promoter expressing GFP in E. coli DH5α are inserted using the promoter probe vector. Were separated, and among them, a vector pbifGFP7 comprising a P G 7 promoter represented by SEQ ID NO: 4 having relatively strong promoter activity and a vector pbifGFP10 comprising P G 10 represented by SEQ ID NO: 5 were obtained. 4 shows a schematic process of producing a promoter probe vector and a schematic cleavage map of the vector pbifGFP7 or pbifGFP10. GFP expression of Escherichia coli comprising the vector pbifGFP7 or pbifGFP10 may be selected by checking green fluorescence by irradiating UV wavelengths. Specifically, the vector pBifGFP7 or pbifGFP10 and the shuttle vector pBES2 according to the present invention were recombinant to prepare a vector pYBGFP7 or pYBGFP10 capable of expressing a gene of interest with the promoter P G 7 or P G 10 according to the present invention. A schematic cleavage map of the vector pYBGFP7 or pYBGFP10 is shown in FIG . 5 .

상기와 같이 대장균 및 비피도박테리움에서 상호 복제되는 셔틀벡터에 목적 유전자를 삽입하여 재조합 벡터를 제조하고, 상기 재조합 벡터를 비피도박테리움에서 발현시킴으로써 목적 유전자 발현시, 별도의 정제 과정 없이도 식품에 첨가할 수 있는 식품 첨가물 또는 경구용 백신의 제조에 사용할 수 있다. 또한, 본 발명에서는 종래에 개발된 대장균 및 비피도박테리움에서 상호 복제되는 셔틀벡터를 이용하여 비피도박테리움을 형질전환시켜 목적 유전자를 발현시킬 때, 형질전환 효율이 매우 낮으면서 목적 유전자가 잘 발현되지 않는 문제점을 해결함으로써 상기 아밀라아제 유전자를 높은 효율로 발현시킬 수 있었다. 구체적으로, 본 발명에서는 셔틀벡터 pBES2의 제조시 사용하였던 플라스미드 pMG1을 분리한 비피도박테리움 롱검 MG1을 아밀라아제 유전자를 삽입한 재조합 벡터 pYBamy59로 형질전환시킴으로써 형질전환체가 전분을 분해하는 것을 확인하였다.As described above, a recombinant vector is prepared by inserting a target gene into a shuttle vector replicated in E. coli and Bifidobacterium, and expressing the recombinant vector in Bifidobacterium. It can be used for the preparation of food additives or oral vaccines which can be added. In addition, in the present invention, when transforming Bifidobacterium to express a target gene using a shuttle vector replicated in the conventionally developed Escherichia coli and Bifidobacterium, the target gene is very low and the transformation efficiency is very low. By solving the problem of not being expressed, the amylase gene could be expressed with high efficiency. Specifically, in the present invention, it was confirmed that the transformant decomposed starch by transforming the Bifidobacterium longgum MG1 from which the plasmid pMG1 used in the preparation of the shuttle vector pBES2 was isolated with the recombinant vector pYBamy59 into which the amylase gene was inserted.

이하, 본 발명을 실시예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail by way of examples.

단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

<실시예 1> 비피도박테리움 롱검 MG1의 분리 및 배양Example 1 Isolation and Culture of Bifidobacterium Longgum MG1

본 발명자들은 한국인의 대장에 서식하는 새로운 유산균 균주를 분리하기 위하여 본 발명자들이 개발한 TP 배지를 사용하여 균주를 분리하였다(한국 특허출원 제 135,780호). 상기 TP 배지는 10 g 트립티케이즈(trypticase), 3.5 g 프로테오스 펩톤 No.3, 3 g 황산 암모니움(ammonium sulfate), 2 g KH2PO4, 1 g K2HPO4, 0.5 g 시스테인·HCl(L-cystein·HCl·H2O), 0.2 g MgSO4, 15 g 한천(agar), 50 ㎖ 트랜스갈락토올리고싸카라이드(transgalactoologosaccharide) 및 50 ㎖ 30% 소디움 프로피오네이트(sodium propionate)로 구성되어 있으며 pH를 7.0으로 조정하여 사용하였다.The present inventors isolated the strain using the TP medium developed by the present inventors to isolate a new lactic acid bacteria strain inhabiting the large intestine of the Korean (Korean Patent Application No. 135,780). The TP medium was 10 g trypticase, 3.5 g proteose peptone No. 3, 3 g ammonium sulfate, 2 g KH 2 PO 4 , 1 g K 2 HPO 4 , 0.5 g cysteine HCl (L-cysteinHClH 2 O), 0.2 g MgSO 4 , 15 g agar, 50 ml transgalactoologosaccharide and 50 ml 30% sodium propionate Consisting of pH was used to adjust the pH to 7.0.

사람의 분변을 0.85% NaCl로 구성된 희석액으로 10-8내지 10-9로 희석한 후 TP 배지에 도말한 후 혐기성 배양기(DIFCO)에서 37℃, 48시간 동안 배양하여 단일 콜로니를 분리하였다. 상기 단일 콜로니를 현미경으로 그 형태학적인 특징을 분석하고, 그람 염색 및 비피더스균 의 고유 효소인 F6PPK(fructose 6 phosphate phosphoketolase) 검사를 수행하여 균주를 동정하였다. 그 결과 형태학적으로는 곤봉형이나 Y자형의 특징이 있었고, 그람 염색 결과 그람 양성균이었으며, K6PPK 활성을 나타내는 비피더스 균임을 확인하였다. 또한, 분리된 균주 중 플라스미드를 함유한 균주만을 분리하였으며, 16S rDNA 염기서열 분석을 통하여 비피도박테리움 롱검 MG1(Bifidobacterium longumMG1)으로 동정되었다.Human feces were diluted to 10 -8 to 10 -9 with a diluent consisting of 0.85% NaCl, then plated in TP medium, and cultured in an anaerobic incubator (DIFCO) for 37 hours and 48 hours to separate single colonies. Strains were identified by analyzing the morphological characteristics of the single colony under a microscope, and by performing Gram staining and F6PPK (fructose 6 phosphate phosphoketolase) tests, which are intrinsic enzymes of Bifidobacteria. As a result, it was morphologically characterized by club-shaped or Y-shaped, Gram-stained, Gram-positive bacteria, Bifidobacteria showing K6PPK activity. In addition, only strains containing plasmids were isolated from the isolates, and identified as Bifidobacterium longum MG1 ( Bifidobacterium longum MG1) through 16S rDNA sequencing.

이와 같이 분리된 균주를 0.05% 시스테인·HCl(L-cysteine·HCl·H2O)이 포함된 BHI 또는 MRS 배지를 이용하여 보관 또는 배양하였다. BHI 배지는 37 g BHI(Difco), 0.01 g 헤민(Hemin), 0.05 g 시스테인 염산, 0.001 g 레사주린(resazurin) 및 0.001 g 비타민 K로 이루어져 있다. 또한, MRS 배지는 18.5 g 글루코스(glucose), 10 g 판크레아틴으로 분해한 젤라틴, 8 g 비프 익스트랙트(beef extract), 4 g 이스트 익스트랙트(yeast extract), 3 g 소디움 아세테이트(sodium acetate), 2 g K2HPO4, 2 g 암모니움 시트레이트(ammonium citrate), 1 g 폴리솔베이트(polysorbate) 80, 0.2 g MgSO4, 0.05 g MnSO4및 0.5 g 시스테인 염산으로 이루어져 있다.The isolated strains were stored or cultured using BHI or MRS medium containing 0.05% cysteine-HCl (L-cysteine-HCl-H 2 O). BHI medium consists of 37 g BHI (Difco), 0.01 g Hemin, 0.05 g cysteine hydrochloric acid, 0.001 g resazurin and 0.001 g vitamin K. MRS medium also contains 18.5 g glucose, 10 g pancreatin digested gelatin, 8 g bee extract, 4 g yeast extract, 3 g sodium acetate, 2 g K 2 HPO 4 , 2 g ammonium citrate, 1 g polysorbate 80, 0.2 g MgSO 4 , 0.05 g MnSO 4 and 0.5 g cysteine hydrochloric acid.

<실시예 2> 비피도박테리움 롱검 MG1으로부터 pMG1의 분리 및 분석Example 2 Isolation and Analysis of pMG1 from Bifidobacterium Longgum MG1

<2-1> 비피도박테리움 롱검 MG1으로부터 pMG1의 분리<2-1> Separation of pMG1 from Bifidobacterium long gum MG1

상기 실시예 1에서 분리·동정된 비피도박테리움 롱검 MG1으로부터 박 등의 방법(Park, M. S.et al.,Letters in Applied Misrobiology, 25:5-7, 1997)을 이용하여서열번호 1로 표시되는 플라스미드 pMG1을 분리하였다.Bifidobacterium long gum isolated and identified in Example 1 is represented by SEQ ID NO: 1 using the method of Park et al. (Park, MS et al. , Letters in Applied Misrobiolog y, 25: 5-7, 1997) The resulting plasmid pMG1 was isolated.

비피도박테리움 롱검 MG1을 0.05% 시스테인·HCl을 포함하는 15-20 ㎖의 MRS 배지를 이용하여 37℃에서 혐기적으로 배양하였다. 원심분리하여 균체를 회수한 후, 다시 30 mM Tris·HCl, 50 mM NaCl, 5 mM EDTA로 구성된 TES(pH 8.0) 용액에서 2회 세척하였다. 상등액을 버린 후 40 mg/㎖의 라이소자임(lysozyme)을 포함하는 5% 수크로스, 50 mM Tris·HCl, 1 mM EDTA를 포함하는 200 ㎕의 수크로스 용액(pH 8.0)을 첨가하고 37℃에서 1 시간 동안 반응하였다. 여기에 3% SDS, 0.2 N NaOH를 포함하는 400 ㎕의 알칼라인 SDS 용액을 첨가한 후, 즉시 15,000 rpm에서 15분간 원심분리하였다. 상등액을 새로운 튜브에 옮기고, 여기에 650 ㎕의 이소프로판올(isopropanol)을 첨가하여 잘 혼합하였다. 이 후, 15,000 rpm에서 15분 동안 원심분리하고 상등액을 제거하여 320㎕의 멸균수에 녹였다. 여기에 200 ㎕의 0.5 ㎎/㎖ EtBr을 포함하는 7.5 M 암모니움 아세테이트(ammonium acetate) 및 350㎕의 페놀/클로로포름(phenol/chloroform)을 첨가하였다. 볼텍싱(vortexing) 한 후 상등액을 새로운 튜브에 옮긴 후 에탄올로 침전하여 정제하였다.Bifidobacterium longgum MG1 was anaerobicly cultured at 37 ° C using 15-20 ml of MRS medium containing 0.05% cysteine-HCl. The cells were recovered by centrifugation, and then washed twice in TES (pH 8.0) solution consisting of 30 mM Tris.HCl, 50 mM NaCl, and 5 mM EDTA. Discard the supernatant and add 200 μl of sucrose solution (pH 8.0) containing 5% sucrose, 40 mM Tris.HCl, 1 mM EDTA containing 40 mg / ml of lysozyme and 1 at 37 ° C. Reacted for hours. To this was added 400 μl alkaline SDS solution containing 3% SDS, 0.2 N NaOH and immediately centrifuged at 15,000 rpm for 15 minutes. The supernatant was transferred to a new tube, to which 650 μl of isopropanol was added and mixed well. Thereafter, the mixture was centrifuged at 15,000 rpm for 15 minutes, the supernatant was removed, and dissolved in 320 µl of sterile water. To this was added 7.5 M ammonium acetate containing 200 μl 0.5 mg / ml EtBr and 350 μl phenol / chloroform. After vortexing, the supernatant was transferred to a new tube and purified by precipitation with ethanol.

<2-2> 비피도박테리움 롱검 MG1으로부터 pMG1의 분석<2-2> Analysis of pMG1 from Bifidobacterium long gum MG1

상기 실시예 2-1에서 분리한 플라스미드 pMG1을 제한효소PvuII,HincII,PstI,NcoI 및SmaI으로 처리하여 제한효소 지도를 작성하고 그 결과를도 1에 나타내었다.Example 2-1 plasmid pMG1 a restriction enzyme isolated from Pvu II, treated with Hinc II, Pst I, Nco I and Sma I creating the restriction map and the results are shown in Figure 1;

또한, pMG1의 전체 염기서열을 결정하기 위하여, pMG1을PvuII으로 처리하고, 대장균 벡터 pUC19(ClonTech)의HincII 위치에 삽입하여 pBES1을 제조하였다. pBES1을 엑소뉴클레아제 III(exonuclease III)로 처리하여 결실 돌연 변이 플라스미드들을 얻은 후 이들을 각각 공지의 방법으로 시퀀싱(sequencing)하여 염기서열을 결정하였다.Further, in order to determine the total base sequence of the pMG1, pMG1 was treated with Pvu II and inserted into Hinc II located in the E. coli vector pUC19 (ClonTech) was prepared pBES1. pBES1 was treated with exonuclease III to obtain deletion mutant plasmids, which were then sequenced by known methods to determine nucleotide sequences.

그 결과 pMG1의 크기는 3,682 bp이고 G+C 함량은 65.1%로 나타났다. 염기서열을 DNASIS(Hitach)로 분석한 결과, 발현이 예상되는 ORFs(open reading frames)가 있었으며 이 중 다른 단백질의 아미노산 서열과 상동성이 높은 서열을 각각 ORF I 및 ORF II라 명명하였다. ORF I은 분자량이 29,000 Da으로 여러 종류의 그람 음성 또는 그람 양성 세균의 복제 단백질(replication protein)과 높은 상동성을 나타냈으며, 그 상부(upstream)에는 22bp 단위로 네 번 반복되는 이테론(iteron)이라 알려진 염기서열이 존재한다. ORF II는 분자량이 71,000 Da으로 다른 세균들의 이동 단백질(mobilization protein)과 높은 서열 상동성을 나타냈으며, 그 상부에는 접합(conjugation)에 중요한 역할을 할 것으로 추정되는ori-T염기 서열이 존재한다. 또한, 상기 ORF I 및 ORF II의 세포 내 발현을 조사하기 위하여 RT-PCR 방법으로 ORF I 및 ORF II를 증폭한 결과 각각 750 bp 및 600 bp의 반응 산물을 확인하였다.As a result, the size of pMG1 was 3,682 bp and G + C content was 65.1%. As a result of analyzing the nucleotide sequence by DNASIS (Hitach), there were ORFs (open reading frames) that are expected to be expressed, and among them, the sequences having high homology with amino acid sequences of other proteins were named ORF I and ORF II, respectively. ORF I has a molecular weight of 29,000 Da and shows high homology with replication proteins of several Gram-negative or Gram-positive bacteria. There is a known base sequence. ORF II showed a high sequence homology with other bacteria's mobilization proteins with a molecular weight of 71,000 Da, and at the top is an ori-T sequence, which is thought to play an important role in conjugation. In addition, the amplification of ORF I and ORF II by RT-PCR method to investigate the expression of the ORF I and ORF II in the cells confirmed the reaction products of 750 bp and 600 bp, respectively.

또한, 각 ORF의 10-15 bp 상부에 라이보좀 결합 부위(ribosome bindingsite)로 예상되는 AGGA 서열이 존재하며, ORF I의 상부에는 22 bp의 염기서열이 4번 반복적으로 나타나는 이테론(iteron) 구조가 존재하며 이러한 구조는 플라스미드의 복제를 조절하는 구조로 알려져 있다. 아울러, ORF II의 상부에는 12 bp로 이루어진ori-T구조가 존재하며 이 구조는 접합에 의해 플라스미드의 전이가 일어나는 기점으로 알려져 있다.In addition, there is an AGGA sequence that is expected to be a ribosome binding site on the top of 10-15 bp of each ORF, and an itron structure in which the 22 bp sequence is repeatedly displayed four times on the top of the ORF I. This structure is known to control the replication of the plasmid. In addition, there is an ori-T structure composed of 12 bp on the top of the ORF II, which is known as the starting point of the transition of the plasmid by conjugation.

<실시예 3> 셔틀벡터 pBES2의 제조Example 3 Preparation of Shuttle Vector pBES2

대장균 및 비피도박테리움에서 상호 복제되는 셔틀벡터를 제조하기 위하여, pUC19(Clontech)에 스타필로코코스(Staphylococcus) 유래의 클로람페니콜 아세틸 트랜스퍼라아제(CAT) 유전자를 가진 pEK104를 제한효소PstI으로 절단한 후, 동일한 제한효소로 절단한 상기 실시예 2-1에서 분리한 pMG1과 연결하여 셔틀벡터 pBES2를 제조하였다.도 6에 상기 pEK104 벡터의 개략적인 개열지도가 도시되어 있다.도 2에 나타낸 바와 같이, 셔틀벡터 pBES2는 pMG1에서 유래한서열번호 2로 표시되는 염기서열을 갖는Mob유전자, 서열번호 3으로 표시되는 염기서열을 갖는Rep유전자 및 pEK104에서 유래한 암피실린 저항성 유전자, CAT 유전자를 포함하고 있으며, 그 크기는 7.8 kb 이다. 상기 셔틀벡터는 30회 정도의 세포분열 후에도 세포 안에서 유지되는 높은 안정성을 보였다.To prepare shuttle vectors that replicate in Escherichia coli and Bifidobacterium, pEK104, a chloramphenicol acetyl transferase (CAT) gene derived from Staphylococcus from pUC19 (Clontech), was digested with restriction enzyme Pst I. Then, the shuttle vector pBES2 was prepared by linking with pMG1 isolated in Example 2-1 cut with the same restriction enzyme. A schematic cleavage map of the pEK104 vector is shown in FIG. 6 . 2, the shuttle vector pBES2 is the ampicillin resistance gene derived from the Rep gene and pEK104 having the nucleotide sequence represented by Mob gene, SEQ ID NO: 3 having the nucleotide sequence shown in SEQ ID NO: 2 derived from pMG1, CAT It contains genes and is 7.8 kb in size. The shuttle vector showed high stability maintained in cells even after about 30 cell divisions.

<실시예 4> 재조합 벡터의 제조 및 발현Example 4 Preparation and Expression of Recombinant Vector

상기 실시예 3에서 제조된 셔틀벡터 pBES2에 목적 유전자를 삽입하고 본 실시예에서 사용한 형질전환 방법을 이용하여 재조합 벡터를 제조하였다.The target vector was inserted into the shuttle vector pBES2 prepared in Example 3, and a recombinant vector was prepared using the transformation method used in this example.

<4-1> 재조합 벡터의 제조<4-1> Preparation of Recombinant Vector

비피도박테리움 롱검 MG1에서 발현시킬 재조합 벡터를 제조하기 위하여, 비피도박테리움 아돌레센티스(B. adolescentis) Int57 유래의 아밀라아제 유전자를 포함하고 있는 pBB3(홍성용, 1991, 고려대학교 석사학위 논문)를XbaI,BamHI으로 각각 절단하였다. 절단한 상기 벡터에 동일한 제한효소로 절단된 상기 실시예 3에서 제조한 셔틀벡터 pBES2를 연결함으로써 재조합 벡터 pYBamy59를 구축하였다. 재조합 벡터 pYBamy59의 개략적인 개열지도와 제조과정이도 3에 도시되어 있다. 상기 재조합 벡터로 대장균 DH5α(Escherichia coli)를 전기천공법으로 형질전환하였다. 상기 형질전환체로부터 발현되는 아밀라아제 활성을 조사하기 위하여, 형질전환체를 1% 전분 및 20 ㎍/㎖ 클로람페니콜이 들어있는 LB 플레이트에 도말하여 37℃에서 24시간 동안 배양하였다. 플레이트 상의 콜로니에 루골 용액(0.5% I2, 5% KI;w/v)을 부어서 콜로니 주위에 투명환이 생기는 것을 확인함으로써 아밀라아제 활성을 확인하였다. 또한, 상기 대장균 형질전환체를 LB 배지에서 배양하고, pYBamy59를 공지의 플라스미드 분리 방법에 따라 분리하여 1.0-1.5 ㎍/㎕으로 농축시킨 후, 비피도박테리움을 형질전환하는데 사용하였다.To prepare a recombinant vector to be expressed in Bifidobacterium longgum MG1, pBB3 (Hong Sung-yong, 1991, Master's dissertation) containing the amylase gene derived from B. adolescentis Int57 was prepared. Each was cut with Xba I and Bam HI. The recombinant vector pYBamy59 was constructed by connecting the shuttle vector pBES2 prepared in Example 3, which was digested with the same restriction enzyme, to the cleaved vector. A schematic cleavage map and manufacturing process of the recombinant vector pYBamy59 is shown in FIG. 3 . E. coli DH5α ( Esherichia coli ) was transformed with the recombinant vector by electroporation. To investigate amylase activity expressed from the transformants, the transformants were plated on LB plates containing 1% starch and 20 μg / ml chloramphenicol and incubated at 37 ° C. for 24 hours. Amylase activity was confirmed by pouring lugol solution (0.5% I2, 5% KI; w / v) into the colonies on the plate to see clear rings around the colonies. In addition, the E. coli transformants were cultured in LB medium, pYBamy59 was isolated according to a known plasmid separation method, concentrated to 1.0-1.5 μg / μl, and used to transform Bifidobacterium.

<4-2> 셔틀벡터를 이용한 비피도박테리움 롱검의 형질전환<4-2> Transformation of Bifidobacterium longgum using shuttle vector

실시예 1에서 분리한 비피도박테리움 롱검 MG1을 0.05%시스테인·염산(cyctein·HCl), 0.5 M 수크로스(sucrose)가 포함된 MRS 배지에 접종하여 37℃에서 혐기성 조건으로 밤샘 배양을 하였다. 상기와 동일한 조성의 배지 250 ㎖에 종배양액을 접종하여 600 nm에서의 흡광도 값이 0.2가 될 때까지 배양하였다. 상기 배양액을 6,000 rpm으로 10 분 동안 4℃에서 원심분리하여 균을 회수하고 회수된 균을 차가운 0.5 M 수크로스 용액으로 현탁하였다. 이와 같이 원심분리와 현탁을 2회 반복하여 컴피턴트 세포(competent cell)를 제조하였다.Bifidobacterium longgum MG1 isolated in Example 1 was inoculated in MRS medium containing 0.05% cysteine hydrochloric acid (cyctein.HCl) and 0.5 M sucrose to incubate overnight at 37 ° C under anaerobic conditions. The seed culture solution was inoculated in 250 ml of the same composition as above, and cultured until the absorbance at 600 nm was 0.2. The culture was centrifuged at 6,000 rpm for 10 minutes at 4 ° C. to recover the bacteria, and the recovered bacteria were suspended in a cold 0.5 M sucrose solution. Thus, centrifugation and suspension were repeated twice to prepare competent cells.

상기에서 제조된 컴피턴트 세포를 셔틀벡터 pBES2로 전기천공법(electroporation)을 이용하여 형질전환하였고, 이 때 옥시레이즈(oxyrase) 효소를 처리함으로써 형질전환 효율을 10배 이상 증가시킬 수 있었다.Competent cells prepared above were transformed with the shuttle vector pBES2 using electroporation, and at this time, the transfection efficiency could be increased by more than 10 times by treating with an oxyrase enzyme.

<4-3> 재조합 벡터의 발현<4-3> Expression of Recombinant Vector

상기 실시예 4-1에서 대장균으로부터 증폭한 pYBamy59로 비피도박테리움 롱검 MG1 및 비피도박테리움 롱검 KJ를 전기천공법으로 형질전환하였다. 상기 형질전환체를 1% 전분 및 4 ㎍/㎖ 클로람페니콜을 포함하는 BHI 배지에서 37℃에서 48시간 동안 배양한 후 실시예 4-1과 같은 방법으로 아밀라아제 활성을 확인하였다. 그 결과 본 발명에서 제조한 재조합 벡터 pYBamy59으로 비피도박테리움이 형질전환된 것을 확인했을 뿐만 아니라 아밀라아제가 비피도박테리움 내에서 높은 효율로 발현되는 것을 확인할 수 있었다.remind Bifidobacterium longgum MG1 and Bifidobacterium longgum KJ were transformed with pYBamy59 amplified from Escherichia coli in Example 4-1 by electroporation. The transformants were incubated for 48 hours at 37 ° C. in BHI medium containing 1% starch and 4 μg / ml chloramphenicol, and amylase activity was confirmed in the same manner as in Example 4-1. As a result, not only did Bifidobacterium be transformed with the recombinant vector pYBamy59 prepared in the present invention, but it was confirmed that amylase was expressed with high efficiency in Bifidobacterium.

<실시예 5> 비피도박테리움 GE65의 프로모터 분리 및 발현Example 5 Promoter Isolation and Expression of Bifidobacterium GE65

비피도박테리움 내에서 목적 유전자를 보다 효과적으로 발현시키기 위하여 비피도박테리움의 프로모터를 분리하여 그 발현 정도를 조사하였다.In order to more effectively express the target gene in Bifidobacterium, the promoter of Bifidobacterium was isolated and its expression level was investigated.

<5-1> 비피도박테리움 GE65의 프로모터 분리<5-1> Promoter Isolation of Bifidobacterium GE65

비피도박테리움의 프로모터를 스크리닝하기 위하여 리포터 유전자로 사용되고 있는 gfp 유전자를 사용하였다.도 4에 나타낸 바와 같이, pEGFP(Clontech)로부터 Plac을 제한효소PvuII 및HincII를 처리하여 제거하여 프로모터 탐침 벡터(promoter probe vector) pEGFP△pro를 제조하였다. 상기 프로모터 탐침 벡터를BamHI으로 절단한 후Sau3AI으로 부분 절단한 비피도박테리움(Bifidobacterium sp.) GE65 염색체 DNA와 연결하였다. 이로부터 대장균 DH5α에서 GFP를 발현하는 프로모터 pbifGFP1, 2, 4, 5, 6, 7, 10을 각각 분리하였고, 이들 중 프로모터 활성이 상대적으로 강한 pbifGFP7 및 10을 얻었다.도 4의 PG1, PG2, PG4, PG5, PG6, PG7, PG10는 pbifGFP에 클로닝된 각각의 프로모터를 나타내며, 화살표의 길이는 프로모터의 길이를 나타내는 것으로서열번호 4로 표시되는 PG7 및서열번호 5로 표시되는 PG10은 각각 그 크기가 228 및 224 bp이다. 상기 pbifGFP를 포함하는 대장균의 GFP 발현은 UV 파장을 조사하여 녹색 형광을 확인함으로써 선별하였다.In order to screen the promoter of Bifidobacterium, the gfp gene, which is used as a reporter gene, was used. 4, was prepared from pEGFP (Clontech) with restriction enzymes Pvu II and P lac Hinc removed by treatment of II with a promoter probe vector (promoter probe vector) pEGFP △ pro as shown in FIG. The promoter probe vector was digested with Bam HI and then linked with Bifidobacterium sp . GE65 chromosomal DNA partially digested with Sau 3AI. From this, promoters pbifGFP1, 2, 4, 5, 6, 7, and 10 expressing GFP in E. coli DH5α were isolated, respectively, and among them, pbifGFP7 and 10 having relatively strong promoter activity were obtained. Fig of 4 P G 1, P G 2 , P G 4, P G 5, P G 6, P G 7, P G 10 denotes the respective promoter cloned into pbifGFP, the length of the arrow represents the length of the promoter P G 7 represented by SEQ ID NO: 4 and P G 10 represented by SEQ ID NO: 5 are 228 and 224 bp in size, respectively. GFP expression of E. coli containing the pbifGFP was selected by checking the green fluorescence by irradiation with UV wavelength.

<5-2> 비피도박테리움 GE65의 프로모터 발현<5-2> Promoter Expression of Bifidobacterium GE65

본 발명에서 제조한 셔틀벡터 pBES2를 제한효소XbaI로 절단한 후, 상기 실시예 5-1에서 분리한 pbifGFP7 또는 10을 동일한 제한효소로 절단하여 얻은 프로모터 PG7 또는 PG10 및gfp유전자를 상기 셔틀벡터 pBES2에 삽입하여, 재조합 벡터 pYBGFP7 또는 pYBGFP10을 제조하였다. 상기 재조합 벡터 pYBGFP7 또는 pYBGFP10의 개략적인 개열지도와 제조과정이도 5에 도시되어 있다. 상기 재조합 벡터 pYBGFP7 또는 pYBGFP10으로 비피도박테리움 롱검 MG1을 전기천공법으로 형질전환하였다. 상기 형질전환체를 MRS 배지에 도말한 후 공기 중에 48시간 노출하고 UV를 조사하여 GFP 단백질의 녹색 형광을 확인함으로써 실시예 5-1에서 분리한 비피도박테리움의 프로모터 활성을 확인할 수 있었다. 또한, RT-PCR 방법(Sambrook, 1989)으로 GFP 단백질의 발현을 전사 단계에서 확인한 결과gfp유전자 크기에 해당하는 400 bp의 밴드를 확인할 수 있었다.Promoters P G 7 or P G 10 and gfp genes obtained by cleaving the shuttle vector pBES2 prepared in the present invention with restriction enzyme Xba I and then cleaving pbifGFP7 or 10 isolated in Example 5-1 with the same restriction enzyme The recombinant vector pYBGFP7 or pYBGFP10 was prepared by inserting into the shuttle vector pBES2. A schematic cleavage map and a manufacturing process of the recombinant vector pYBGFP7 or pYBGFP10 are shown in FIG . 5 . Bifidobacterium longgum MG1 was transformed with the recombinant vector pYBGFP7 or pYBGFP10 by electroporation. The transformant was smeared in MRS medium, and then exposed to air for 48 hours and irradiated with UV to confirm the green fluorescence of the GFP protein, thereby confirming the promoter activity of the Bifidobacterium isolated in Example 5-1. In addition, as a result of confirming the expression of the GFP protein in the transcription step by RT-PCR method (Sambrook, 1989), it was confirmed that the band of 400 bp corresponding to the size of the gfp gene.

상기에서 살펴본 바와 같이, 본 발명자들은 대장균 및 비피도박테리움에서 상호 복제가 가능하면서 목적 유전자를 효율적으로 발현시킬 수 있는 셔틀벡터를 개발하고 비피도박테리움 내에서 목적 유전자를 강하게 발현시키는 프로모터를 분리하였다. 본 발명의 셔틀벡터 및 프로모터를 이용하면 목적 유전자를 발현시 별도의 정제 과정이 필요 없으며, 비피도박테리움 내에서 발현된 목적 유전자를 바로 식품에 첨가할 수 있어 식품 첨가물 또는 경구용 백신의 제조에 사용될 수 있는 장점이 있다. 또한, 이러한 셔틀벡터 개발을 통하여 비피도박테리움을 이용한 프로바이오틱스의 잠재력과 가능성을 획기적으로 높일 수 있다.As described above, the present inventors have developed a shuttle vector capable of efficiently replicating a gene of interest in Escherichia coli and Bifidobacterium, and isolates a promoter that strongly expresses the gene of interest in Bifidobacterium. It was. By using the shuttle vector and the promoter of the present invention, there is no need for a separate purification process when the target gene is expressed, and the target gene expressed in Bifidobacterium can be added directly to food for the preparation of food additives or oral vaccines. There is an advantage that can be used. In addition, the development of such a shuttle vector can significantly increase the potential and potential of probiotics using Bifidobacterium.

<110> BIFIDO INC. <120> Shuttle vector comprising a novel plasmid originated from Bifidobacterium longum MG1 <130> NP04-0080 <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 3681 <212> DNA <213> Bifidobacterium longum MG1(KFCC-11273) <400> 1 gcagatcaag tccctgcagg agaggcaggc ccagctcaag gcccgcgaga acgacctcat 60 ggcgcggcgc agggaacgcg aacgcagggc ccgcaccaag cgcctgatcg aggtcggcgc 120 gatggccgag tcggccgcgg gcttcgaggg cggcgacgag agggccaagg agcgcatagc 180 ccgcctcgtg cagctcggct cgctggtgga agccatgtgc tccaccgacg tgatggacaa 240 ctacacgagc cgcgaggacc tcaagaccac cgtcaccaag gccctggaac acaacgtcag 300 aaccagcgat ggcatgaact ggaacctgca cgacctggtg tacgaggcgc tgagcgagga 360 atggggcaga agggacggcg agatcagcga cctctgggcg gacgacgggc caagcggata 420 ccagccaccc tcatacgagc cggtcaaccc cgaacgcagg actccccaaa caccctccga 480 tggcctgatc tgacgtccaa aaaaaggcgc cgtgcgccct ttttaaatct tttaaaatct 540 ttttacattc ttttaggccc tccgcagccc ttggaaacat tgggctcaga ggatgttact 600 ggggacaaaa agggagcgaa ccggggacaa aaagggagcg aaccggggac aaaaagggag 660 cgaaccgggg acaaaaaggg agcgaaccgg ggacgttgct aaaatgtgtc tcctttttga 720 tcaaggtggg gactcaaatt atttgtggac taacttaatt tgagtccccc ataggagcta 780 tgctaaggcc atgtccaatg agatcgtgaa gttcagcaac cagttcaaca acgtggcact 840 gaagaagttc gacgccgtgc acctggacgt gctcatggcg atcgcctcaa gggtgaggga 900 gaagggcacg gccacggtgg agttctcgtt cgaggagctg cgcggcctca tgcggctgag 960 gaagaacctg accaacaggc agctggccga caagatcgtg cagacgaacg cgcggctgct 1020 ggcgttgaac tacatgttcg aggattcggg caagatcatc cagttcgcgc tgttcacgaa 1080 gttcgttacc gacccgcagg aggcgaccct cgcggttggg gtcaacgagg agttcgcgtt 1140 cctgctcaac gacctgacca gccagttcac gcgcttcgag ctggccgagt tcgccgacct 1200 caaaagcaag tacgccaagg agttctaccg cagggccaaa caataccgca gctccggaat 1260 ctggaagatc agccgcgacg agttctgccg actgctcagc gttcccaaat ccacagccga 1320 gcaagtgaga gatctcaaca aacgagtcct caagccgatt atcgaggagt gtgggccact 1380 ccttggactg aagatcgagc gccagtacgt gaaacgcagg ctgtcggggt tcgtgttcac 1440 gttcgcccgc gagacccctc cggtgatcga cgccaggccc gtggaggcga ggaaggcgga 1500 ggatgcgggc cattggacga agcgtcgccg ggtacggcga ggtgttcacg accactgagc 1560 tgttcgacgt gacggccgcg cgtgaccact tcgacggcac cgtggaggcc ggggaatgcc 1620 gtttctgcgc gtttgacgct cgcaaccgcg aacatcatgc gcagaacgcc ggaaagctgt 1680 tctagcggcc gtgtccgcgc cttggggcgg ttgcgcgctc catgggtcga tctgccgctg 1740 ttcgcgcctc ccgctggcct gtgagcctgt ccgtgcgctg tctgatctcg ttgagcaggt 1800 cggccttggt cctgggggcc tggcgtgatt cgaacgggct ggcctctccc cagtcctcgg 1860 gctcgctgag gtccagcggc tcgtcgccgg acggtgcggg ccgtttcgtg tcctgcgtcg 1920 ggttctccgc ctgcgcgcgt tgttcggcca tacgcagtgc gagggccttc acctgttcgg 1980 ggcttggccg ttcgctctcg gccgttcggc ctgttcgagc cgcctctcca gttcggccac 2040 gacgcctggt cctcggctgc atgtcgtggt cgtagatggc cttggtggtc ctcatgcgga 2100 acctgttggc ctggtcccag tcggccggga tgtcggcgtc ttcgagccac ggcaccgccc 2160 cgcgcagcct ctcggactgc tctccggcca cctgctcggc gttccgcagc agcctgccgc 2220 gcttgaagat gttcgcgtcc ctggcctcgc gcgaggccgc ccgcgccacg tcgatgatct 2280 gcttggcggc ctcgtgctcc tcggtgcccg tggcttctcc accacttctc ccggttcccc 2340 acgtcggccc tcgcgtccat gagttttccg cggatcgcgg ccgtgtggtc ggccttgtcg 2400 gcttcgagct ggcggcgcgc gtcgccctcg aagtagcgcc agttcggctc ggccggttcc 2460 ggccgcgcct ggcgtcggcg cagccgcccg atcttggcgg cgaacagctc gcccaggcgg 2520 tcgaatatcc gtccgagttc ggcgcggacg gcggtcagca ggctgttgct gcgcctgatc 2580 tcacggttgg tctggcacct ctcgctgacg cctccggccc gttcgatggc cctggcggcg 2640 tatccctcgt ggatggtggg ttcgaggtcg ctgccctggt cttcgaggct cctgtggtcg 2700 attctcgcgg tctcgtccag ccgcgcgttg caggtgttcg cccaggactc gcgcagggcc 2760 ttgagcttgg ccttccggtc gagcgggttc agggacacgc tcgtgcgctt ccactgcctg 2820 cggccccgct tgtcggtctt ctgcctgccg gtctcggggt cgatcagcgg aacccgctcg 2880 ccccgctcgt ccagcacgta ctccatgcgc tgcttgagcc gggcccagcc gcccgtggcc 2940 gggtcgatct gccggttggc gacgaggatg tgcgcgtgcg ggttgttgcc ccgcccgtcg 3000 tcgtggatgg cgtaggtggc cgcgtagccg tccgcgttca ggttctcgcg gatgtactcc 3060 tccagcgcct gcacgcgctg cctgggggtg aactcgcgcg gcagggccac gacgatcttc 3120 ttggccggcc tcgccgtccg gccggtctcg tgcagctcga ccgcgttgaa cagcacggcg 3180 gggtcggcgt actcggccgg cgcgccctcc ggcagcaggg tgccgacgcg cagcacgtct 3240 cgctccttgc gcccgtagtc gtaggcgccg cgacgctcgt catgcacccg cctgccgctg 3300 atgtacgaga gcgtggccgt ggccttggaa ccgctcgcgc ggctcacgtt ggagacggac 3360 agatggtaga tcgccatcgg cttcggctcc tttcgtgatc ggcgcgggcg gcggggtgcg 3420 gggcctcggc cctgcggcaa ggggttccca ggggtgcggc gagcaccccc gggcctgccg 3480 ggaggctccc ccggaagggt gggaatccaa agggcaacgc ccgtggcccc cggagggcgc 3540 gcttacggaa aatgcaacct ccggttgcat gtaagtgcgc cctaatcttt gattagggat 3600 tccttgctgg tagaatcata tcaccatacg gatgatgcag accatgtaag gagccgtttc 3660 gatggtgaag agcctggatg a 3681 <210> 2 <211> 1941 <212> DNA <213> pMG1(KFCC-11273) <400> 2 atggcgatct accatctgtc cgtctccaac gtgagccgcg cgagcggttc caaggccacg 60 gccacgctct cgtacatcag cggcaggcgg gtgcatgacg agcgtcgcgg cgcctacgac 120 tacgggcgca aggagcgaga cgtgctgcgc gtcggcaccc tgctgccgga gggcgcgccg 180 gccgagtacg ccgaccccgc cgtgctgttc aacgcggtcg agctgcacga gaccggccgg 240 acggcgaggc cggccaagaa gatcgtcgtg gccctgccgc gcgagttcac ccccaggcag 300 cgcgtgcagg cgctggagga gtacatccgc gagaacctga acgcggacgg ctacgcggcc 360 acctacgcca tccacgacga cgggcggggc aacaacccgc acgcgcacat cctcgtcgcc 420 aaccggcaga tcgacccggc cacgggcggc tgggcccggc tcaagcagcg catggagtac 480 gtgctggacg agcggggcga gcgggttccg ctgatcgacc ccgagaccgg caggcagaag 540 accgacaagc ggggccgcag gcagtggaag cgcacgagcg tgtccctgaa cccgctcgac 600 cggaaggcca agctcaaggc cctgcgcgag tcctgggcga acacctgcaa cgcgcggctg 660 gacgagaccg cgagaatcga ccacaggagc ctcgaagacc agggcagcga cctcgaaccc 720 accatccacg agggatacgc cgccagggcc atcgaacggg ccggaggcgt cagcgagagg 780 tgccagacca accgtgagat caggcgcagc aacagcctgc tgaccgccgt ccgcgccgaa 840 ctcggacgga tattcgaccg cctgggcgag ctgttcgccg ccaagatcgg gcggctgcgc 900 cgacgccagg cgcggccgga accggccgag ccgaactggc gctacttcga gggcgacgcg 960 cgccgccagc tcgaagccga caaggccgac cacacggccg cgatccgcgg aaaactcatg 1020 gacgcgaggg ccgacgtggg gaaccgggag aagtggtgga gaagccacgg gcaccgagga 1080 gcacgaggcc gccaagcaga tcatcgacgt ggcgcgggcg gcctcgcgcg aggccaggga 1140 cgcgaacatc ttcaagcgcg gcaggctgct gcggaacgcc gagcaggtgg ccggagagca 1200 gtccgagagg ctgcgcgggg cggtgccgtg gctcgaagac gccgacatcc cggccgactg 1260 ggaccaggcc aacaggttcc gcatgaggac caccaaggcc atctacgacc acgacatgca 1320 gccgaggacc aggcgtcgtg gccgaactgg agaggcggct cgaacaggcc gaacggccga 1380 gagcgaacgg ccaagccccg aacaggtgaa ggccctcgca ctgcgtatgg ccgaacaacg 1440 cgcgcaggcg gagaacccga cgcaggacac gaaacggccc gcaccgtccg gcgacgagcc 1500 gctggacctc agcgagcccg aggactgggg agaggccagc ccgttcgaat cacgccaggc 1560 ccccaggacc aaggccgacc tgctcaacga gatcagacag cgcacggaca ggctcacagg 1620 ccagcgggag gcgcgaacag cggcagatcg acccatggag cgcgcaaccg ccccaaggcg 1680 cggacacggc cgctagaaca gctttccggc gttctgcgca tgatgttcgc ggttgcgagc 1740 gtcaaacgcg cagaaacggc attccccggc ctccacggtg ccgtcgaagt ggtcacgcgc 1800 ggccgtcacg tcgaacagct cagtggtcgt gaacacctcg ccgtacccgg cgacgcttcg 1860 tccaatggcc cgcatcctcc gccttcctcg cctccacggg cctggcgtcg atcaccggag 1920 gggtctcgcg ggcgaacgtg a 1941 <210> 3 <211> 798 <212> DNA <213> pMG1(KFCC-11273) <400> 3 atgtccaatg agatcgtgaa gttcagcaac cagttcaaca acgtggcact gaagaagttc 60 gacgccgtgc acctggacgt gctcatggcg atcgcctcaa gggtgaggga gaagggcacg 120 gccacggtgg agttctcgtt cgaggagctg cgcggcctca tgcggctgag gaagaacctg 180 accaacaggc agctggccga caagatcgtg cagacgaacg cgcggctgct ggcgttgaac 240 tacatgttcg aggattcggg caagatcatc cagttcgcgc tgttcacgaa gttcgttacc 300 gacccgcagg aggcgaccct cgcggttggg gtcaacgagg agttcgcgtt cctgctcaac 360 gacctgacca gccagttcac gcgcttcgag ctggccgagt tcgccgacct caaaagcaag 420 tacgccaagg agttctaccg cagggccaaa caataccgca gctccggaat ctggaagatc 480 agccgcgacg agttctgccg actgctcagc gttcccaaat ccacagccga gcaagtgaga 540 gatctcaaca aacgagtcct caagccgatt atcgaggagt gtgggccact ccttggactg 600 aagatcgagc gccagtacgt gaaacgcagg ctgtcggggt tcgtgttcac gttcgcccgc 660 gagacccctc cggtgatcga cgccaggccc gtggaggcga ggaaggcgga ggatgcgggc 720 cattggacga agcgtcgccg ggtacggcga ggtgttcacg accactgagc tgttcgacgt 780 gacggccgcg cgtgacca 798 <210> 4 <211> 228 <212> DNA <213> Bifidobacterium sp. <400> 4 cagaaaattt caacttagct gattatctag ttcctacttt ggcttttatt cttgtactag 60 ttgtaatttt gctctttaaa gtcaactcag gacgtttcta taatcctttt gtataaaagt 120 ttcttttcct gccaagataa gttaaaatat actaataaaa agaaagaggg agagctttat 180 gaataacttt tcacaagaac cagaacgccg tacaattgtc gacgtaac 228 <210> 5 <211> 223 <212> DNA <213> Bifidobacterium sp. <400> 5 ggggatcctc tagaggatcg tccacacttn cctgatgaag tagcattagg agtaacttac 60 aaagatgttg ccgatcactt agaaggtaaa gatgtgagtg aagaagcagc tgaaccgatt 120 gnaaagttgt ggaaaaagag tgaacataag cgtcatttgc cagtaactat ttttgatgat 180 ttctataagc aaaattagtt aagtattttt cttggaggaa ata 223<110> BIFIDO INC. <120> Shuttle vector comprises a novel plasmid originated from          Bifidobacterium longum MG1 <130> NP04-0080 <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 3681 <212> DNA <213> Bifidobacterium longum MG1 (KFCC-11273) <400> 1 gcagatcaag tccctgcagg agaggcaggc ccagctcaag gcccgcgaga acgacctcat 60 ggcgcggcgc agggaacgcg aacgcagggc ccgcaccaag cgcctgatcg aggtcggcgc 120 gatggccgag tcggccgcgg gcttcgaggg cggcgacgag agggccaagg agcgcatagc 180 ccgcctcgtg cagctcggct cgctggtgga agccatgtgc tccaccgacg tgatggacaa 240 ctacacgagc cgcgaggacc tcaagaccac cgtcaccaag gccctggaac acaacgtcag 300 aaccagcgat ggcatgaact ggaacctgca cgacctggtg tacgaggcgc tgagcgagga 360 atggggcaga agggacggcg agatcagcga cctctgggcg gacgacgggc caagcggata 420 ccagccaccc tcatacgagc cggtcaaccc cgaacgcagg actccccaaa caccctccga 480 tggcctgatc tgacgtccaa aaaaaggcgc cgtgcgccct ttttaaatct tttaaaatct 540 ttttacattc ttttaggccc tccgcagccc ttggaaacat tgggctcaga ggatgttact 600 ggggacaaaa agggagcgaa ccggggacaa aaagggagcg aaccggggac aaaaagggag 660 cgaaccgggg acaaaaaggg agcgaaccgg ggacgttgct aaaatgtgtc tcctttttga 720 tcaaggtggg gactcaaatt atttgtggac taacttaatt tgagtccccc ataggagcta 780 tgctaaggcc atgtccaatg agatcgtgaa gttcagcaac cagttcaaca acgtggcact 840 gaagaagttc gacgccgtgc acctggacgt gctcatggcg atcgcctcaa gggtgaggga 900 gaagggcacg gccacggtgg agttctcgtt cgaggagctg cgcggcctca tgcggctgag 960 gaagaacctg accaacaggc agctggccga caagatcgtg cagacgaacg cgcggctgct 1020 ggcgttgaac tacatgttcg aggattcggg caagatcatc cagttcgcgc tgttcacgaa 1080 gttcgttacc gacccgcagg aggcgaccct cgcggttggg gtcaacgagg agttcgcgtt 1140 cctgctcaac gacctgacca gccagttcac gcgcttcgag ctggccgagt tcgccgacct 1200 caaaagcaag tacgccaagg agttctaccg cagggccaaa caataccgca gctccggaat 1260 ctggaagatc agccgcgacg agttctgccg actgctcagc gttcccaaat ccacagccga 1320 gcaagtgaga gatctcaaca aacgagtcct caagccgatt atcgaggagt gtgggccact 1380 ccttggactg aagatcgagc gccagtacgt gaaacgcagg ctgtcggggt tcgtgttcac 1440 gttcgcccgc gagacccctc cggtgatcga cgccaggccc gtggaggcga ggaaggcgga 1500 ggatgcgggc cattggacga agcgtcgccg ggtacggcga ggtgttcacg accactgagc 1560 tgttcgacgt gacggccgcg cgtgaccact tcgacggcac cgtggaggcc ggggaatgcc 1620 gtttctgcgc gtttgacgct cgcaaccgcg aacatcatgc gcagaacgcc ggaaagctgt 1680 tctagcggcc gtgtccgcgc cttggggcgg ttgcgcgctc catgggtcga tctgccgctg 1740 ttcgcgcctc ccgctggcct gtgagcctgt ccgtgcgctg tctgatctcg ttgagcaggt 1800 cggccttggt cctgggggcc tggcgtgatt cgaacgggct ggcctctccc cagtcctcgg 1860 gctcgctgag gtccagcggc tcgtcgccgg acggtgcggg ccgtttcgtg tcctgcgtcg 1920 ggttctccgc ctgcgcgcgt tgttcggcca tacgcagtgc gagggccttc acctgttcgg 1980 ggcttggccg ttcgctctcg gccgttcggc ctgttcgagc cgcctctcca gttcggccac 2040 gacgcctggt cctcggctgc atgtcgtggt cgtagatggc cttggtggtc ctcatgcgga 2100 acctgttggc ctggtcccag tcggccggga tgtcggcgtc ttcgagccac ggcaccgccc 2160 cgcgcagcct ctcggactgc tctccggcca cctgctcggc gttccgcagc agcctgccgc 2220 gcttgaagat gttcgcgtcc ctggcctcgc gcgaggccgc ccgcgccacg tcgatgatct 2280 gcttggcggc ctcgtgctcc tcggtgcccg tggcttctcc accacttctc ccggttcccc 2340 acgtcggccc tcgcgtccat gagttttccg cggatcgcgg ccgtgtggtc ggccttgtcg 2400 gcttcgagct ggcggcgcgc gtcgccctcg aagtagcgcc agttcggctc ggccggttcc 2460 ggccgcgcct ggcgtcggcg cagccgcccg atcttggcgg cgaacagctc gcccaggcgg 2520 tcgaatatcc gtccgagttc ggcgcggacg gcggtcagca ggctgttgct gcgcctgatc 2580 tcacggttgg tctggcacct ctcgctgacg cctccggccc gttcgatggc cctggcggcg 2640 tatccctcgt ggatggtggg ttcgaggtcg ctgccctggt cttcgaggct cctgtggtcg 2700 attctcgcgg tctcgtccag ccgcgcgttg caggtgttcg cccaggactc gcgcagggcc 2760 ttgagcttgg ccttccggtc gagcgggttc agggacacgc tcgtgcgctt ccactgcctg 2820 cggccccgct tgtcggtctt ctgcctgccg gtctcggggt cgatcagcgg aacccgctcg 2880 ccccgctcgt ccagcacgta ctccatgcgc tgcttgagcc gggcccagcc gcccgtggcc 2940 gggtcgatct gccggttggc gacgaggatg tgcgcgtgcg ggttgttgcc ccgcccgtcg 3000 tcgtggatgg cgtaggtggc cgcgtagccg tccgcgttca ggttctcgcg gatgtactcc 3060 tccagcgcct gcacgcgctg cctgggggtg aactcgcgcg gcagggccac gacgatcttc 3120 ttggccggcc tcgccgtccg gccggtctcg tgcagctcga ccgcgttgaa cagcacggcg 3180 gggtcggcgt actcggccgg cgcgccctcc ggcagcaggg tgccgacgcg cagcacgtct 3240 cgctccttgc gcccgtagtc gtaggcgccg cgacgctcgt catgcacccg cctgccgctg 3300 atgtacgaga gcgtggccgt ggccttggaa ccgctcgcgc ggctcacgtt ggagacggac 3360 agatggtaga tcgccatcgg cttcggctcc tttcgtgatc ggcgcgggcg gcggggtgcg 3420 gggcctcggc cctgcggcaa ggggttccca ggggtgcggc gagcaccccc gggcctgccg 3480 ggaggctccc ccggaagggt gggaatccaa agggcaacgc ccgtggcccc cggagggcgc 3540 gcttacggaa aatgcaacct ccggttgcat gtaagtgcgc cctaatcttt gattagggat 3600 tccttgctgg tagaatcata tcaccatacg gatgatgcag accatgtaag gagccgtttc 3660 gatggtgaag agcctggatg a 3681 <210> 2 <211> 1941 <212> DNA <213> pMG1 (KFCC-11273) <400> 2 atggcgatct accatctgtc cgtctccaac gtgagccgcg cgagcggttc caaggccacg 60 gccacgctct cgtacatcag cggcaggcgg gtgcatgacg agcgtcgcgg cgcctacgac 120 tacgggcgca aggagcgaga cgtgctgcgc gtcggcaccc tgctgccgga gggcgcgccg 180 gccgagtacg ccgaccccgc cgtgctgttc aacgcggtcg agctgcacga gaccggccgg 240 acggcgaggc cggccaagaa gatcgtcgtg gccctgccgc gcgagttcac ccccaggcag 300 cgcgtgcagg cgctggagga gtacatccgc gagaacctga acgcggacgg ctacgcggcc 360 acctacgcca tccacgacga cgggcggggc aacaacccgc acgcgcacat cctcgtcgcc 420 aaccggcaga tcgacccggc cacgggcggc tgggcccggc tcaagcagcg catggagtac 480 gtgctggacg agcggggcga gcgggttccg ctgatcgacc ccgagaccgg caggcagaag 540 accgacaagc ggggccgcag gcagtggaag cgcacgagcg tgtccctgaa cccgctcgac 600 cggaaggcca agctcaaggc cctgcgcgag tcctgggcga acacctgcaa cgcgcggctg 660 gacgagaccg cgagaatcga ccacaggagc ctcgaagacc agggcagcga cctcgaaccc 720 accatccacg agggatacgc cgccagggcc atcgaacggg ccggaggcgt cagcgagagg 780 tgccagacca accgtgagat caggcgcagc aacagcctgc tgaccgccgt ccgcgccgaa 840 ctcggacgga tattcgaccg cctgggcgag ctgttcgccg ccaagatcgg gcggctgcgc 900 cgacgccagg cgcggccgga accggccgag ccgaactggc gctacttcga gggcgacgcg 960 cgccgccagc tcgaagccga caaggccgac cacacggccg cgatccgcgg aaaactcatg 1020 gacgcgaggg ccgacgtggg gaaccgggag aagtggtgga gaagccacgg gcaccgagga 1080 gcacgaggcc gccaagcaga tcatcgacgt ggcgcgggcg gcctcgcgcg aggccaggga 1140 cgcgaacatc ttcaagcgcg gcaggctgct gcggaacgcc gagcaggtgg ccggagagca 1200 gtccgagagg ctgcgcgggg cggtgccgtg gctcgaagac gccgacatcc cggccgactg 1260 ggaccaggcc aacaggttcc gcatgaggac caccaaggcc atctacgacc acgacatgca 1320 gccgaggacc aggcgtcgtg gccgaactgg agaggcggct cgaacaggcc gaacggccga 1380 gagcgaacgg ccaagccccg aacaggtgaa ggccctcgca ctgcgtatgg ccgaacaacg 1440 cgcgcaggcg gagaacccga cgcaggacac gaaacggccc gcaccgtccg gcgacgagcc 1500 gctggacctc agcgagcccg aggactgggg agaggccagc ccgttcgaat cacgccaggc 1560 ccccaggacc aaggccgacc tgctcaacga gatcagacag cgcacggaca ggctcacagg 1620 ccagcgggag gcgcgaacag cggcagatcg acccatggag cgcgcaaccg ccccaaggcg 1680 cggacacggc cgctagaaca gctttccggc gttctgcgca tgatgttcgc ggttgcgagc 1740 gtcaaacgcg cagaaacggc attccccggc ctccacggtg ccgtcgaagt ggtcacgcgc 1800 ggccgtcacg tcgaacagct cagtggtcgt gaacacctcg ccgtacccgg cgacgcttcg 1860 tccaatggcc cgcatcctcc gccttcctcg cctccacggg cctggcgtcg atcaccggag 1920 gggtctcgcg ggcgaacgtg a 1941 <210> 3 <211> 798 <212> DNA <213> pMG1 (KFCC-11273) <400> 3 atgtccaatg agatcgtgaa gttcagcaac cagttcaaca acgtggcact gaagaagttc 60 gacgccgtgc acctggacgt gctcatggcg atcgcctcaa gggtgaggga gaagggcacg 120 gccacggtgg agttctcgtt cgaggagctg cgcggcctca tgcggctgag gaagaacctg 180 accaacaggc agctggccga caagatcgtg cagacgaacg cgcggctgct ggcgttgaac 240 tacatgttcg aggattcggg caagatcatc cagttcgcgc tgttcacgaa gttcgttacc 300 gacccgcagg aggcgaccct cgcggttggg gtcaacgagg agttcgcgtt cctgctcaac 360 gacctgacca gccagttcac gcgcttcgag ctggccgagt tcgccgacct caaaagcaag 420 tacgccaagg agttctaccg cagggccaaa caataccgca gctccggaat ctggaagatc 480 agccgcgacg agttctgccg actgctcagc gttcccaaat ccacagccga gcaagtgaga 540 gatctcaaca aacgagtcct caagccgatt atcgaggagt gtgggccact ccttggactg 600 aagatcgagc gccagtacgt gaaacgcagg ctgtcggggt tcgtgttcac gttcgcccgc 660 gagacccctc cggtgatcga cgccaggccc gtggaggcga ggaaggcgga ggatgcgggc 720 cattggacga agcgtcgccg ggtacggcga ggtgttcacg accactgagc tgttcgacgt 780 gacggccgcg cgtgacca 798 <210> 4 <211> 228 <212> DNA <213> Bifidobacterium sp. <400> 4 cagaaaattt caacttagct gattatctag ttcctacttt ggcttttatt cttgtactag 60 ttgtaatttt gctctttaaa gtcaactcag gacgtttcta taatcctttt gtataaaagt 120 ttcttttcct gccaagataa gttaaaatat actaataaaa agaaagaggg agagctttat 180 gaataacttt tcacaagaac cagaacgccg tacaattgtc gacgtaac 228 <210> 5 <211> 223 <212> DNA <213> Bifidobacterium sp. <400> 5 ggggatcctc tagaggatcg tccacacttn cctgatgaag tagcattagg agtaacttac 60 aaagatgttg ccgatcactt agaaggtaaa gatgtgagtg aagaagcagc tgaaccgatt 120 gnaaagttgt ggaaaaagag tgaacataag cgtcatttgc cagtaactat ttttgatgat 180 ttctataagc aaaattagtt aagtattttt cttggaggaa ata 223

Claims (17)

서열번호 1로 표시되는 염기서열을 갖는 플라스미드 pMG1과 대장균 유래 형질전환용 벡터를 연결하여 제조된, 대장균 및 비피도박테리움에서 상호 복제가 가능한 셔틀벡터.A shuttle vector capable of mutually replicating in Escherichia coli and Bifidobacterium prepared by linking a plasmid pMG1 having a nucleotide sequence represented by SEQ ID NO: 1 with an E. coli-derived transformation vector. 제1항에 있어서, 상기 플라스미드 pMG1은 비피도박테리움 롱검 MG1(Bifidobacterium longumMG1)(기탁번호 KFCC-11273)에서 유래된 것을 특징으로 하는 셔틀벡터.The shuttle vector according to claim 1, wherein the plasmid pMG1 is derived from Bifidobacterium longum MG1 (Accession No. KFCC-11273). 제1항 또는 제2항에 있어서, 상기 플라스미드 pMG1에서 유래된 서열번호 2로 표시되는 염기서열을 갖는Mob유전자, 상기 플라스미드 pMG1에서 유래된 서열번호 3으로 표시되는 염기서열을 갖는Rep유전자 및 선별 유전자를 포함하는 것을 특징으로 하는 셔틀벡터.The Mob gene having a nucleotide sequence represented by SEQ ID NO: 2 derived from the plasmid pMG1, a Rep gene having a nucleotide sequence represented by SEQ ID NO: 3 derived from the plasmid pMG1, and a selection gene according to claim 1 or 2 Shuttle vector comprising a. 제1항에 있어서, 상기 대장균 유래 형질전환용 벡터가 pEK104, pUC19 및 pBR322로 이루어진 군에서 선택되는 어느 하나인 것을 특징으로 하는 셔틀벡터.The shuttle vector according to claim 1, wherein the E. coli-derived transformation vector is any one selected from the group consisting of pEK104, pUC19, and pBR322. 제4항에 있어서, 플라스미드 pMG1과 pEK104를 연결하여 제조된, 도 2에 나타낸 개열지도를 갖는 셔틀벡터.The shuttle vector according to claim 4, having a cleavage map shown in FIG. 2, prepared by linking plasmids pMG1 and pEK104. 제3항에 있어서, 선별 유전자는 암피실린(ampicillin) 저항성 유전자, 가나마이신(kanamycin) 저항성 유전자 및 클로람페니콜 아세틸 트랜스퍼라아제(chloramphenicol acetyl transferase) 유전자로 이루어진 군에서 선택되는 하나 이상인 것을 특징으로 하는 셔틀벡터.The shuttle vector of claim 3, wherein the selection gene is at least one selected from the group consisting of ampicillin resistance gene, kanamycin resistance gene, and chloramphenicol acetyl transferase gene. 제1항의 셔틀벡터에 목적 단백질을 코딩하는 목적 유전자를 삽입한 재조합 벡터.A recombinant vector in which a target gene encoding a target protein is inserted into the shuttle vector of claim 1. 제7항에 있어서, 상기 목적 유전자는 아밀라아제 유전자, 백신유전자, 항암유전자 및 다양한 생리활성을 나타낼 수 있는 유전자로 이루어진 군으로부터 선택되는 어느 하나인 것을 특징으로 하는 재조합 벡터.The recombinant vector according to claim 7, wherein the gene of interest is any one selected from the group consisting of amylase gene, vaccine gene, anticancer gene and gene capable of exhibiting various physiological activities. 제8항에 있어서, 상기 재조합 벡터가 목적 유전자로서 아밀라아제 유전자를 가지며 도 3에 나타낸 개열지도를 갖는 pYBamy59인 것을 특징으로 하는 재조합 벡터.The recombinant vector according to claim 8, wherein the recombinant vector is pYBamy59 having an amylase gene as a target gene and having a cleavage map shown in FIG. 제7항의 재조합 벡터로 형질전환된 비피도박테리움 속 미생물.A microorganism of the genus Bifidobacterium transformed with the recombinant vector of claim 7. 제10항에 있어서, 제9항의 재조합 벡터 pYBamy59로 형질전환된 비피도박테리움 롱검 MG1(기탁번호 KFCC-11274).The Bifidobacterium longgum MG1 according to claim 10 transformed with the recombinant vector pYBamy59 of claim 9 (Accession No. KFCC-11274). 제1항 또는 제7항의 벡터에 서열번호 4로 표시되는 염기서열을 갖는 비피도박테리움 GE65(Bifidobacterium sp. GE65)의 프로모터를 삽입한 재조합 벡터.A recombinant vector in which the promoter of Bifidobacterium GE65 ( Bifidobacterium sp . GE65) having a nucleotide sequence represented by SEQ ID NO: 4 is inserted into a vector of claim 1 or 7. 제12항에 있어서, 제5항의 셔틀벡터에 서열번호 4로 표시되는 염기서열을 갖는 비피도박테리움 GE65의 프로모터를 삽입한 재조합 벡터 pYBGFP7.The recombinant vector pYBGFP7 according to claim 12, wherein a promoter of Bifidobacterium GE65 having a nucleotide sequence represented by SEQ ID NO: 4 is inserted into the shuttle vector of claim 5. 제12항의 재조합 벡터로 형질전환된 비피도박테리움 속 미생물.A microorganism of the genus Bifidobacterium transformed with the recombinant vector of claim 12. 제1항 또는 제7항의 벡터에 서열번호 5로 표시되는 염기서열을 갖는 비피도박테리움 GE65의 프로모터를 삽입한 재조합 벡터.A recombinant vector having a promoter of Bifidobacterium GE65 having a nucleotide sequence represented by SEQ ID NO: 5 inserted into a vector according to claim 1. 제15항에 있어서, 제5항의 셔틀벡터에 서열번호 5로 표시되는 염기서열을 갖는 비피도박테리움 GE65의 프로모터를 삽입한 재조합 벡터 pYBGFP10.The recombinant vector pYBGFP10 according to claim 15, wherein a promoter of Bifidobacterium GE65 having a nucleotide sequence represented by SEQ ID NO: 5 is inserted into the shuttle vector of claim 5. 제15항의 재조합 벡터로 형질전환된 비피도박테리움 속 미생물.A microorganism of the genus Bifidobacterium transformed with the recombinant vector of claim 15.
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KR100715730B1 (en) * 2005-11-15 2007-05-08 연세대학교 산학협력단 24A novel Bifidobacterium longum A24 with anti-bacterial activity against toxigenic pathogens
KR102343938B1 (en) * 2020-09-02 2021-12-28 주식회사 비피도 Composition for improving inflammation containing recombinant Bifidobacterium bifidum BGN4

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KR101039596B1 (en) 2008-08-20 2011-06-09 건국대학교 산학협력단 A novel method for expressing protein in vivo

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100715730B1 (en) * 2005-11-15 2007-05-08 연세대학교 산학협력단 24A novel Bifidobacterium longum A24 with anti-bacterial activity against toxigenic pathogens
KR102343938B1 (en) * 2020-09-02 2021-12-28 주식회사 비피도 Composition for improving inflammation containing recombinant Bifidobacterium bifidum BGN4

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