KR100988164B1

KR100988164B1 - Production of the antigenic domain recombinant protein of Helicobacter pylori Cag26 reactive to human antisera and its method

Info

Publication number: KR100988164B1
Application number: KR1020080007194A
Authority: KR
Inventors: 조명제; 전진수; 이우곤; 윤희상; 서지현; 이광호; 최상한; 송재영; 백승철; 강형련; 권영철
Original assignee: 경상대학교산학협력단
Priority date: 2008-01-23
Filing date: 2008-01-23
Publication date: 2010-10-18
Also published as: KR20090081224A

Abstract

본 발명은 헬리코박터 파이로리 감염의 혈청학적 진단을 목적으로 헬리코박터 파이로리(Helicobacter pylori)가 생성하는 Cag26 단백질 중 사람에게서 항원성을 보이는 항원도메인만 생성하는 헬리코박터 파이로리 Cag26 단백 항원도메인재조합 단백질 제조방법에 관한 것이다.The present invention relates to a method for preparing a Helicobacter pylori Cag26 protein antigen domain recombination protein that generates only an antigenic domain of human antigen among Cag26 proteins produced by Helicobacter pylori for the purpose of serological diagnosis of Helicobacter pylori infection.

이차원전기영동과 면역블롯 분석법으로 헬리코박터 파이로리 항원도메인을 맵핑(mapping)하여 CagA 단백질 중 환자의 혈청과 강하게 반응하는 수용성인 항원도메인재조합 단백질을 제조함으로써, 혈청학적 진단을 위한 항원 성분의 이용을 용이하게 하는 장점이 있다. Two-dimensional electrophoresis and immunoblot analysis map the Helicobacter pylori antigen domain to produce a water-soluble antigen domain recombination protein that reacts strongly with the patient's serum in the CagA protein, thereby facilitating the use of antigen components for serological diagnosis. There is an advantage.

헬리코박터 파이로리, 항원도메인재조합 단백질, Cag26 단백질, 혈청학적 진단 Helicobacter pylori, antigen domain recombination protein, Cag26 protein, serological diagnosis

Description

헬리코박터 파이로리의 Ｃａｇ26 단백질의 사람 항혈청에 반응하는 항원도메인재조합 단백질 및 그의 제조방법{Production of the antigenic domain recombinant protein of Helicobacter pylori Cag26 reactive to human antisera and its method} Production domain of the antigenic domain recombinant protein of Helicobacter pylori Cag26 reactive to human antisera and its method

본 발명은 감염환자의 혈청과 반응하는 헬리코박터 파이로리 Cag26 단백질의 항원도메인재조합 단백질과 그의 제조방법에 관한 것으로서, 헬리코박터 파이로리 감염 여부 진단을 목적으로 헬리코박터 파이로리가 생성하는 Cag26 단백 중 사람에게서 항원성을 보이고 수용성으로 대장균에서 발현되는 항원도메인만 생성하는 헬리코박터 파이로리 Cag26 단백질 항원도메인재조합 단백질 및 그의 제조방법에 관한 것이다. The present invention relates to an antigen-domain recombination protein of Helicobacter pylori Cag26 protein that reacts with the serum of an infected patient and a method for preparing the same. The present invention relates to a Helicobacter pylori Cag26 protein antigen domain recombination protein which produces only an antigen domain expressed in E. coli and a method for producing the same.

헬리코박터 파이로리 감염의 진단은 크게 침습적 방법과 비침습적 방법으로 나눈다. 침습적 방법는 위 내시경술을 통하여 얻은 생검체를 이용하여 배양법, 현미경적 관찰, 요소분해효소측정 등 헬리코박터 파이로리 세균의 존재를 확인하는 방법이다. 침습적 방법은 육안으로 병변을 관찰하면서 위점막을 채취하기 때문에 높은 특이도를 갖는다는 장점이 있으나, 헬리코박터 파이로리가 위점막에 골고루 분포하는 것이 아니고 군데군데 서식하기 때문에 채취하는 위점막 위치에 따라 그 결과가 달리 나올 수가 있다. 또한 환자에게 신체적인 고통을 주기 때문에 반복 검사를 시행하기가 어려우므로 현실적으로 약물 투여 후 효과판정으로 사용하기가 불가능하다. 비침습적 방법은 ¹³C-요소호흡법과 혈청학적 방법이 있다. ¹³C-요소호흡법은 반복 시행할 수 있다는 장점이 있으나 검사하는데 고가의 장비가 필요하고 감염균수가 적을 경우 위 음성으로 진단된다. 또한 인지능력이 부족한 소아의 경우는 진단에 사용하기가 쉽지 않다. 혈청학적 방법은 모든 의료기관에서 널리 사용하고 있기 때문에 인적ㆍ물적 자원이 준비되어 있어 간편하게 진단할 수 있는 장점이 있다. 그러나 사용하는 항원에 따라서 교차반응이 일어날 수 있고 현증환자뿐만 아니라 과거에 감염된 경우에도 양성반응이 나올 수가 있다. 그러나 이러한 문제점은 진단에 사용하는 항원을 새로이 개발함으로써 극복할 수 있다. Diagnosis of Helicobacter pylori infection is largely divided into invasive and non-invasive methods. Invasive methods are Helicobacter pylori such as culture method, microscopic observation, urease measurement using biopsy specimen obtained through gastroscopy. This is how you check the presence of bacteria. The invasive method has the advantage of having high specificity because the gastric mucosa is collected while observing the lesions with the naked eye.However, because Helicobacter pylori is not evenly distributed in the gastric mucosa, it is inhabited in several places. Can come out differently. In addition, since it is difficult to perform repeated tests because of physical pain to the patient, it is practically impossible to use it as an effect test after drug administration. Non-invasive methods include ¹³ C-urea breathing and serological methods. ^{13 The} C-urea breathing method has the advantage of being repeated, but if the test requires expensive equipment and the number of infected bacteria is small, the diagnosis is negative. In addition, children with cognitive abilities are not easy to use for diagnosis. Since serological methods are widely used in all medical institutions, human and physical resources are prepared, which makes it easy to diagnose them. However, depending on the antigen used, cross reactions may occur and positive reactions may occur in the past as well as in patients with dizziness. However, this problem can be overcome by developing new antigens for diagnosis.

헬리코박터 파이로리가 감염된 환자의 혈청은 다양한 종류의 헬리코박터 파이로리 세포단백질과 반응하는데 특히 120 ~ 140 kDa 크기의 단백질과 특이하게 반응한다고 알려져 있다. 이 단백질은 세포독소생산과 관련이 있다고 하여 Cag26 단백으로 명명되었다. 서양에서는 혈청학적 조사에 의하면 십이지장궤양환자의 80 ~ 100%가 항Cag26 항체 양성인데 비해 무증상 보균자의 경우 60%만이 항Cag26 항체 양성자이다. 위암이 호발하는 한국뿐만 아니라 일본과 중국에서는 만성위염 환자에서 분리된 균주와 위ㆍ십이지장궤양 환자에서 분리되는 균주는 모두 Cag26 단백질 생산균주이고 모두 Cag26에 대한 항체를 보유한다. 상기 단백질은 사람에게서 항원성이 강하다. Serum from patients infected with Helicobacter pylori is known to react specifically with a variety of Helicobacter pylori cell proteins, specifically with proteins ranging in size from 120 to 140 kDa. The protein is named Cag26 protein because it is involved in cytotoxin production. In the West, serological studies indicate that 80-100% of duodenal ulcer patients are anti-Cag26 antibody-positive, whereas only 60% of asymptomatic carriers are anti-Cag26 antibody-positive. Strains isolated from patients with chronic gastritis and those isolated from patients with gastric and duodenal ulcers in Korea, as well as in Korea, where gastric cancer occurs, are Cag26 protein-producing strains, and both have antibodies against Cag26. The protein is highly antigenic in humans.

일반적으로 단백항원의 경우 아미노산 15 ~ 22 잔기가 한 개의 항원결정기를 형성한다고 알려져 있다. 경우에 따라서는 이러한 항원결정기 여러 개가 항원분자의 일정 부분에 모여 항원도메인을 형성한다. 따라서 항원분자 모든 부분이 항원성을 나타내기보다 특정한 위치의 도메인이 항원성이 높은 구조를 나타낸다. 이러한 항체 반응성이 높은 항원도메인을 분석하고 이를 유전자 재조합기법으로 생산하고 정제하여 항원으로 사용함으로써 항원단백질 전체를 항원으로 사용하는 경우보다 더욱 특이적인 진단이 가능할 것으로 사료된다. In the case of protein antigens, amino acid 15-22 residues are generally known to form one epitope. In some cases, several of these epitopes are collected in a certain portion of the antigen molecule to form an antigen domain. Therefore, rather than all of the antigen molecules show antigenicity, the domain at a specific position shows a high antigenic structure. By analyzing these antigenic domains with high antibody reactivity, producing them, refining them, and using them as antigens, it is possible to make more specific diagnosis than when using whole antigen proteins as antigens.

그러나 종래에는 Cag26 단백 전체를 유전자 재조합기법으로 대장균을 이용하여 발현을 성공하였다는 보고가 없으며, 특히 Cag26 단백은 분자의 크기가 크고 또한 헬리코박터 파이로리 자체에서도 발현양이 적어 정제하기가 쉽지 않아, 정제된 Cag26 단백을 혈청학적 진단법에 사용하는 것이 매우 제한적이다. However, there have been no reports of successful expression of E. coli using the entire recombinant protein of Cag26. In particular, Cag26 protein has a large molecule size and a small amount of expression in Helicobacter pylori itself. The use of Cag26 protein in serological diagnostics is very limited.

본 발명은 헬리코박터 파이로리의 Cag26 단백질의 사람 항혈청에 반응하는 항원도메인재조합 단백질 및 그의 제조방법에 관한 것으로써, 헬리코박터 파이로리의 주된 항원성분인 Cag26 단백을 분석하여 환자혈청과 반응하는 항원도메인 부분을 결정하고 반응성과 용해성이 확인된 항원도메인재조합 단백질을 발현하고 정제하는 방법을 제공하여 혈청학적 진단법에 적용시키고자 한다. 보다 상세하게는 헬리코박터 파이로리 Cag26 단백 유전자 절편을 중복되게 나누어 클로닝하고 발현시 켜 환자혈청과의 반응성을 분석한다. 상기와 같이 항원도메인이 위치하는 곳을 매핍(mapping)함으로써 환자혈청과 반응하는 항원도메인 부분을 확인한다. 수용성인 항원도메인재조합 단백질을 발현시켜 정제함으로써, 본 발명을 완성하였다. The present invention relates to an antigen domain recombination protein that reacts to human antiserum of Cag26 protein of Helicobacter pylori, and to a method for preparing the same, wherein the antigen domain that reacts with patient serum is determined by analyzing Cag26 protein, which is a major antigen component of Helicobacter pylori. The present invention provides a method for expressing and purifying antigen domain recombination proteins that have been confirmed to be reactive and soluble. More specifically, Helicobacter pylori Cag26 protein fragments were divided into duplicates, cloned and expressed to analyze their reactivity with patient serum. As described above, the antigen domain portion that reacts with the patient serum is identified by mapping the place where the antigen domain is located. The present invention was completed by expressing and purifying a water-soluble antigen domain recombination protein.

따라서 본 발명의 제 1목적은 헬리코박터 파이로리의 Cag26 단백 항원도메인를 제공하는 것이다. Therefore, the first object of the present invention is to provide a Cag26 protein antigen domain of Helicobacter pylori.

따라서 본 발명의 제 2목적은 상기 항원도메인를 포함하는 수용성인 헬리코박터 파이로리의 Cag26 단백 항원도메인재조합 단백질을 제공하는 것이다.Therefore, a second object of the present invention is to provide a water-soluble Cag26 protein antigen domain recombination protein of Helicobacter pylori containing the antigen domain.

또한, 본 발명의 제 3목적은 수용성인 헬리코박터 파이로리 Cag26 단백 항원도메인재조합 단백질의 제조방법을 제공하는 것이다.It is also a third object of the present invention to provide a method for producing a water-soluble Helicobacter pylori Cag26 protein antigen domain recombination protein.

상기와 같은 목적을 해결하기 위하여, 본 발명은 수용성인 헬리코박터 파이로리 Cag26 단백 항원도메인재조합 단백질 및 그의 제조방법을 제공하고자 한다. In order to solve the above object, the present invention is to provide a water-soluble Helicobacter pylori Cag26 protein antigen domain recombination protein and a method for producing the same.

본 발명은 하기의 서열번호 40으로 표시되는 아미노산 서열로 구성되는 헬리코박터 파이로리의 Cag26 단백 항원도메인에 관한 것이다. 본 발명에서 확인한 헬리코박터 파이로리의 Cag26 단백 중 사람에게서 항원도메인으로 작용하는 부위의 아미노산 서열은 다음과 같다. The present invention relates to a Cag26 protein antigen domain of Helicobacter pylori composed of the amino acid sequence represented by SEQ ID NO: 40 below. Among the Cag26 proteins of Helicobacter pylori identified in the present invention, the amino acid sequence of a site acting as an antigen domain in humans is as follows.

EASKEARAAAFDPNLKGIRSELSDKLENINKNLKDFGKSFDELKNGKNKDFSKAEETLKALKDSVKDLGINEASKEARAAAFDPNLKGIRSELSDKLENINKNLKDFGKSFDELKNGKNKDFSKAEETLKALKDSVKDLGIN

PEWISKIENLNAALNDFKNGKNKDFSKVTQAKSDLENSIKDVIINQKITDKVDNLNQAVSET PEWISKIENLNAALNDFKNGKNKDFSKVTQAKSDLENSIKDVIINQKITDKVDNLNQAVSET

(서열번호 40)(SEQ ID NO 40)

또한, 본 발명은 상기의 아미노산 서열을 갖는 항원도메인을 포함하는 헬리코박터 파이로리의 Cag26 단백 항원도메인재조합 단백질에 관한 것이다. 상기 헬리코박터 파이로리 Cag26 단백 항원도메인은 Cag26 단백 유전자의 염기서열 1999번에서 2397번까지의 도메인에 위치한다.The present invention also relates to a Cag26 protein antigen domain recombination protein of Helicobacter pylori comprising an antigen domain having the above amino acid sequence. The Helicobacter pylori Cag26 protein antigen domain is located in the nucleotide sequence of the Cag26 protein gene from 1999 to 2397 domain.

또한, 본 발명은 서열번호 41의 아미노산 서열로 구성되는 헬리코박터 파이로리의 Cag26 단백 항원도메인재조합 단백질에 관한 것이다. 상기 서열번호 41의 아미노산 서열은 다음과 같다.The present invention also relates to a Cag26 protein antigen domain recombination protein of Helicobacter pylori composed of the amino acid sequence of SEQ ID 41. The amino acid sequence of SEQ ID 41 is as follows.

MAKEIKFSDSARNLLFEGVRQLHDAVKVTMGPRGRNVLIQKSYGAPSITKDGVSVAKEIELSCPVANMGAQMAKEIKFSDSARNLLFEGVRQLHDAVKVTMGPRGRNVLIQKSYGAPSITKDGVSVAKEIELSCPVANMGAQ

LVKEVASKTADAAGDGTTTATVLAYSIFKEGLRNITAGANPIEVKRGMDKAAEAIINELKKASKKVGGKEELVKEVASKTADAAGDGTTTATVLAYSIFKEGLRNITAGANPIEVKRGMDKAAEAIINELKKASKKVGGKEE

ITQVATISANSDHNIGKLIADAMEKVGKDGVITVEEAKGIEDELDVVEGMQFDRGYLSPYFVTNAEKMTAQITQVATISANSDHNIGKLIADAMEKVGKDGVITVEEAKGIEDELDVVEGMQFDRGYLSPYFVTNAEKMTAQ

LDNAYILLTDKKISSMKDILPLLEKTMKEGKPLLIIAEDIEGEAEASKEARAAAFDPNLKGIRSELSDKLELDNAYILLTDKKISSMKDILPLLEKTMKEGKPLLIIAEDIEGEAEASKEARAAAFDPNLKGIRSELSDKLE

NINKNLKDFGKSFDELKNGKNKDFSKAEETLKALKDSVKDLGINPEWISKIENLNAALNDFKNGKNKDFSKNINKNLKDFGKSFDELKNGKNKDFSKAEETLKALKDSVKDLGINPEWISKIENLNAALNDFKNGKNKDFSK

VTQAKSDLENSIKDVIINQKITDKVDNLNQAVSET (서열번호 41)VTQAKSDLENSIKDVIINQKITDKVDNLNQAVSET (SEQ ID NO: 41)

상기 단백질은 헬리코박터 파이로리 감염환자의 항혈청과 반응하는 수용성 헬리코박터 파이로리 Cag26 단백 항원도메인재조합 단백질이다.The protein is a water soluble Helicobacter pylori Cag26 protein antigen domain recombination protein that reacts with the antiserum of a Helicobacter pylori infected patient.

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

이 때, 사용되는 기술 용어 및 과학 용어에 있어서 다른 정의가 없다면, 이 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 통상적으로 이해하고 있는 의미를 가진다.At this time, if there is no other definition in the technical terms and scientific terms used, it has a meaning commonly understood by those of ordinary skill in the art.

또한, 종래와 동일한 기술적 구성 및 작용에 대한 반복되는 설명은 생략하기로 한다.Repeated descriptions of the same technical constitution and operation as those of the conventional art will be omitted.

본 발명은,The present invention,

1) 헬리코박터 파이로리 항원 유전자 절편을 증폭하는 단계;1) amplifying the Helicobacter pylori antigen gene fragment;

2) 상기 증폭된 유전자 절편을 서브-클로닝 하여 클론을 얻은 후, 제한효소를 처리하여 절단하는 단계;2) sub-cloning the amplified gene fragment to obtain a clone, and then digesting and digesting the restriction enzyme;

3) 상기 절단된 유전자 절편을 분리한 후, 정제하는 단계;3) separating the purified gene fragment and then purifying;

4) 상기 2) 단계에서 처리한 동일한 제한효소를 벡터에 처리하는 단계; 4) treating the vector with the same restriction enzyme treated in step 2);

5) 상기 3) 단계의 정제된 유전자 절편과 상기 4) 단계의 벡터를 클로닝 하여 클론을 얻는 단계; 5) cloning the purified gene fragment of step 3) and the vector of step 4) to obtain a clone;

6) 상기 5) 단계의 클론을 단백질 발현시켜 항원성을 조사하고 항원도메인 서열을 맵핑하는 단계;6) protein expression of the clone of step 5) to investigate antigenicity and to map the antigen domain sequence;

7) 상기 6) 단계의 맵핑된 항원도메인 서열을 증폭하고 융합대상 벡터와 클로닝 하여 클론을 얻는 단계; 및7) amplifying the mapped antigen domain sequence of step 6) and cloning with a fusion target vector to obtain a clone; And

8) 상기 7) 단계의 클론을 발현하여 재조합 융합단백질을 정제하는 단계:8) Purifying the recombinant fusion protein by expressing the clone of step 7):

를 포함하는 헬리코박터 파이로리 Cag26 단백 항원도메인재조합 단백질의 제조방법에 관한 것이다.Helicobacter pylori Cag26 protein antigen domain comprising a method for producing a recombinant protein comprising a.

상기 1) 단계, 2) 단계, 또는 4) 단계에서 헬리코박터 파이로리 발현용 벡터 pEGexs에 융합단백질로 발현되도록 코돈을 맞추기 위해서는 5’말단에는 EcoRI을 사용하며, 3’말단은 XhoI을 사용한다. Eco RI is used at the 5 'end, and Xho I is used at the 3' end to adjust the codon to be expressed as a fusion protein in the pEGexs for the Helicobacter pylori expression vector in step 1), 2), or 4).

본 발명에서는 상기의 헬리코박터 파이로리 Cag26 유전자 절편의 크기를 약 200 ~ 600 bp로 나누어 헬리코박터 파이로리 발현용 벡터 pEGexs에 클로닝 하여 재조합 단백질을 생산하고 이를 환자혈청과 반응시켜 항원도메인을 맵핑(mapping)하였다. 융합단백질의 반응성과 용해성을 조사하여 환자혈청과 반응성이 높으면서 용해성으로 발현되는 Cag26 단백 항원도메인을 포함하는 재조합 융합단백질을 제조하고 정제함으로써 혈청학적 진단법에 광범위하게 사용될 수 있게 되었다. In the present invention, the size of the Helicobacter pylori Cag26 gene fragment was divided into about 200 to 600 bp and cloned into Helicobacter pylori expression vector pEGexs to produce a recombinant protein and reacted with the patient serum to map the antigen domain. By investigating the reactivity and solubility of the fusion protein, a recombinant fusion protein comprising a Cag26 protein antigen domain which is highly reactive with patient serum and is expressed in soluble form was prepared and purified, and thus can be widely used in serological diagnostic methods.

이상과 같이, 본 발명은 환자혈청에 반응하는 수용성 헬리코박터 파이로리 Cag26 단백 항원도메인재조합 단백질 및 그의 제조방법을 제공함으로써, 헬리코박터 파이로리 혈청학적 진단에 사용할 수 있는 새로운 항원 성분을 제공하여 헬리코박터 파이로리의 혈청학적 진단법 개발의 폭을 넓힘으로써 위십이지장 질환의 보건관리를 보다 향상시킬 수 있다.As described above, the present invention provides a water-soluble Helicobacter pylori Cag26 protein antigen domain recombination protein and a method for preparing the same, which provides a novel antigen component that can be used for serological diagnosis of Helicobacter pylori by responding to patient serum. By broadening the development, the health management of gastroduodenal diseases can be improved.

이하, 본 발명을 구체적인 실시 예에 의해 보다 더 상세히 설명하고자 한다. 하지만, 본 발명은 하기 실시 예에 의해 한정되는 것은 아니며, 본 발명의 사상과 범위 내에서 여러 가지 변형 또는 수정할 수 있음은 이 분야에서 당업자에게 명백한 것이다.Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited by the following examples, and various modifications or changes can be made within the spirit and scope of the present invention to those skilled in the art.

[실시예 1] 헬리코박터 파이로리의 Cag26 단백 유전자분석Example 1 Cag26 Protein Gene Analysis of Helicobacter Pylori

국내에서 분리된 헬리코박터 파이로리 Cag26 단백 유전자 염기서열 자료를 분석한 결과, 헬리코박터 파이로리 Cag26 단백 유전자 서열은 하기 서열번호 1과 같이 전체크기는 3,534 bp이고 1,177개 아미노산으로 형성되어 있다. 또한 하기 도 1에 나타낸 바와 같이, 염기서열 분석 프로그램인 레이저진(Lasergene)으로 헬리코박터 파이로리 Cag26 단백 유전자를 분석하여 염기서열 183, 1456, 2449 번째에는 제한효소 HindIII 자리, 548 번째에는 Pvul 자리, 1026 번째에는 PstI 자리, 1644 번째에는 SmaI 자리, 2022 번째에는 NotI 자리, 2235 번째에 SphI 자리가 위치하고 있는 확인할 수 있었다.As a result of analyzing the Helicobacter pylori Cag26 protein gene sequence data isolated in Korea, the Helicobacter Pylori Cag26 protein gene sequence is 3,534 bp in total size as shown in SEQ ID NO: 1 and is formed of 1,177 amino acids. In addition, as shown in Figure 1, by analyzing the Helicobacter pylori Cag26 protein gene with a laser sequencing program (Lasergene) nucleotide sequence 183, 1456, 2449th restriction enzyme Hind III site, 548th Pvu l site, The 1026 th position was identified as the Pst I position, the 1644 th Sma I position, the 2022 th Not Not I position, and the 2235 th Sph I position.

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cgcattaatagcaatgtccaaactggagcgatcaatgaaaaagcgaccggcatgctaacg 3240cgcattaatagcaatgtccaaactggagcgatcaatgaaaaagcgaccggcatgctaacg 3240

caaaaaaaccctgagtggctcaagctcgtgaatgataagatagttgcacataatgtggga 3300 caaaaaaaccctgagtggctcaagctcgtgaatgataagatagttgcacataatgtggga 3300

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(서열번호 1)(SEQ ID NO 1)

[제조예 1] 헬리코박터 파이로리 항원 제조 및 결손변이항원 제조Preparation Example 1 Preparation of Helicobacter Pylori Antigen and Deletion of Mutant Antigen

헬리코박터 파이로리 Cag26 단백은 전체 유전자가 클로닝 되어도 전체 단백이 발현되지 않으므로, 상기 실시예 1의 자료를 근거로 하여 유전자 크기를 약 400 bp씩 중복으로 나누어 17개의 절편을 증폭하고 이를 헬리코박터 파이로리 발현용 벡터 pEGexs에 클로닝 하였다. Since Helicobacter pylori Cag26 protein is not expressed even when the entire gene is cloned, amplifying 17 fragments by dividing the gene size by about 400 bp in duplicate based on the data of Example 1, and expressing the vector pEGexs for Helicobacter pylori expression vector. Cloned into.

우선, Cag26 단백을 발현하기 위하여 본 발명에서 사용한 프라이머 세트의 서열은 다음과 같다.First, the sequence of the primer set used in the present invention to express the Cag26 protein is as follows.

Cag26-1Cag26-1

순방향 프라이머 : 5’-GCACCGGAATTCATGACTAATGAAACC-3’(서열번호 2)Forward primer: 5'-GCACCGGAATTCATGACTAATGAAACC-3 '(SEQ ID NO: 2)

역방향 프라이머 : 5’-GCGTCGCTCGAGATCTTTTTGAAGAGA-3’(서열번호 3)Reverse primer: 5'-GCGTCGCTCGAGATCTTTTTGAAGAGA-3 '(SEQ ID NO: 3)

Cag26-2Cag26-2

순방향 프라이머 : 5’-GCACCGGAATTCTTCACTCTTGGTGAT- 3’(서열번호 4)Forward primer: 5'-GCACCGGAATTCTTCACTCTTGGTGAT-3 '(SEQ ID NO: 4)

역방향 프라이머 : 5’-GCGTCGCTCGAGCATGAAATCTACTTT- 3’(서열번호 5)Reverse primer: 5'-GCGTCGCTCGAGCATGAAATCTACTTT-3 '(SEQ ID NO: 5)

Cag26-3 Cag26-3

순방향 프라이머 : 5’-GCACCGGAATTCGATTTCATGGAATTTC- 3’(서열번호 6)Forward primer: 5'-GCACCGGAATTCGATTTCATGGAATTTC- 3 '(SEQ ID NO: 6)

역방향 프라이머 : 5’-GCATAGCTCGAGGCTACTTTGCTAAAA- 3’(서열번호 7)Reverse primer: 5'-GCATAGCTCGAGGCTACTTTGCTAAAA-3 '(SEQ ID NO: 7)

Cag26-4 Cag26-4

순방향 프라이머 : 5’-GCACCGGAATTCAAAGATCCGTCTAAA - 3’(서열번호 8)Forward primer: 5'-GCACCGGAATTCAAAGATCCGTCTAAA-3 '(SEQ ID NO: 8)

역방향 프라이머 : 5’-GCATAACTCGAGATCACCAAGAGTGAA - 3’(서열번호 9)Reverse primer: 5'-GCATAACTCGAGATCACCAAGAGTGAA-3 '(SEQ ID NO: 9)

Cag26-5 Cag26-5

순방향 프라이머 : 5’-GCACAGGAATTCACTACGAATGGCGTT- 3’(서열번호 10)Forward primer: 5'-GCACAGGAATTCACTACGAATGGCGTT-3 '(SEQ ID NO: 10)

역방향 프라이머 : 5’-GCATAACTCGAGGTCGTTTCTGCTCT- 3’(서열번호 11)Reverse primer: 5'-GCATAACTCGAGGTCGTTTCTGCTCT-3 '(SEQ ID NO: 11)

Cag26-6 Cag26-6

순방향 프라이머 : 5’-GCAATGGAATTCGAGGCTAGTAAGGAA- 3’(서열번호 12)Forward primer: 5'-GCAATGGAATTCGAGGCTAGTAAGGAA-3 '(SEQ ID NO: 12)

역방향 프라이머 : 5’-GCATAACTCGAGAGTCTCTGATACAG- 3’(서열번호 13)Reverse primer: 5'-GCATAACTCGAGAGTCTCTGATACAG-3 '(SEQ ID NO: 13)

Cag26-7Cag26-7

순방향 프라이머 : 5’-GCCCCGAATTCAAATTAACAGGCGAT- 3’(서열번호 14)Forward primer: 5'-GCCCCGAATTCAAATTAACAGGCGAT-3 '(SEQ ID NO: 14)

역방향 프라이머 : 5’-GCCACCTCGAGAATTTTGTTAATCCG - 3’(서열번호 15)Reverse primer: 5'-GCCACCTCGAGAATTTTGTTAATCCG-3 '(SEQ ID NO: 15)

Cag26-8Cag26-8

순방향 프라이머 : 5’-GCCACGAATTCGCATCAGCAGGTAAA- 3’(서열번호 16)Forward primer: 5'-GCCACGAATTCGCATCAGCAGGTAAA- 3 '(SEQ ID NO: 16)

역방향 프라이머 : 5’-GCCATCTCGAGTCCAGTTTGGACATT- 3’(서열번호 17)Reverse primer: 5'-GCCATCTCGAGTCCAGTTTGGACATT-3 '(SEQ ID NO: 17)

Cag26-9Cag26-9

순방향 프라이머 : 5’-GCCATGAATTCCAAACTGGAGCGATC- 3’(서열번호 18)Forward primer: 5'-GCCATGAATTCCAAACTGGAGCGATC-3 '(SEQ ID NO: 18)

역방향 프라이머 : 5’-GCCATCTCGAGTTTTTGGAAACCACC- 3’(서열번호 19)Reverse primer: 5'-GCCATCTCGAGTTTTTGGAAACCACC-3 '(SEQ ID NO: 19)

Cag26-10Cag26-10

순방향 프라이머 : 5’-GCACCGGAATTCAGGCAAGCTTTTGAG- 3’(서열번호 20)Forward primer: 5'-GCACCGGAATTCAGGCAAGCTTTTGAG- 3 '(SEQ ID NO: 20)

역방향 프라이머 : 5’-GCCATCCTCGAGCCTTGCTTTCAAAGA- 3’(서열번호 21)Reverse primer: 5'-GCCATCCTCGAGCCTTGCTTTCAAAGA-3 '(SEQ ID NO: 21)

Cag26-11Cag26-11

순방향 프라이머 : 5’-GCCACGAATTCAGGCAAGAAGCAGAA- 3’(서열번호 22)Forward primer: 5'-GCCACGAATTCAGGCAAGAAGCAGAA-3 '(SEQ ID NO: 22)

역방향 프라이머 : 5’-GCCTACTCGAGAACCTTTTCAGGTTC- 3’(서열번호 23)Reverse primer: 5'-GCCTACTCGAGAACCTTTTCAGGTTC-3 '(SEQ ID NO: 23)

Cag26-12Cag26-12

순방향 프라이머 : 5’-GCCCCGAATTCTATGGGGATAATGGT- 3’(서열번호 24)Forward primer: 5'-GCCCCGAATTCTATGGGGATAATGGT-3 '(SEQ ID NO: 24)

역방향 프라이머 : 5’-GCCACCTCGAGAACCAAAGCTAAATG- 3’(서열번호 25)Reverse primer: 5'-GCCACCTCGAGAACCAAAGCTAAATG-3 '(SEQ ID NO: 25)

Cag26-13Cag26-13

순방향 프라이머 : 5’-GCCACGAATTCGAGTTTGGTAATGGG- 3’(서열번호 26)Forward primer: 5'-GCCACGAATTCGAGTTTGGTAATGGG-3 '(SEQ ID NO: 26)

역방향 프라이머 : 5’-GCCCCCTCGAGTGAAACTTTTCTAAC- 3’(서열번호 27)Reverse primer: 5'-GCCCCCTCGAGTGAAACTTTTCTAAC-3 '(SEQ ID NO: 27)

Cag26-14Cag26-14

순방향 프라이머 : 5’-GCCCCGAATTCCAATAAAGCTGTAGC- 3’(서열번호 28)Forward primer: 5'-GCCCCGAATTCCAATAAAGCTGTAGC-3 '(SEQ ID NO: 28)

역방향 프라이머 : 5’-GCATACTCGAGCACCGAGTCTTTAAG- 3’(서열번호 29)Reverse primer: 5'-GCATACTCGAGCACCGAGTCTTTAAG-3 '(SEQ ID NO: 29)

Cag26-15Cag26-15

순방향 프라이머 : 5’-GCCCCGAATTCGTGAAAGATTTAGGC - 3’(서열번호 30)Forward primer: 5'-GCCCCGAATTCGTGAAAGATTTAGGC-3 '(SEQ ID NO: 30)

역방향 프라이머 : 5’-GCCCCCTCGAGCAATTCTTTCCTGAT- 3’(서열번호 31)Reverse primer: 5'-GCCCCCTCGAGCAATTCTTTCCTGAT-3 '(SEQ ID NO: 31)

Cag26-16Cag26-16

순방향 프라이머 : 5’-GCCCCGAATTCAAAGAATTGAACGAG- 3’(서열번호 32)Forward primer: 5'-GCCCCGAATTCAAAGAATTGAACGAG- 3 '(SEQ ID NO: 32)

역방향 프라이머 : 5’-GCATACTCGAGTTCTTGTTCCCTTGA- 3’(서열번호 33)Reverse primer: 5'-GCATACTCGAGTTCTTGTTCCCTTGA-3 '(SEQ ID NO: 33)

Cag26-17Cag26-17

순방향 프라이머 : 5’-GCCACGAATTCCGTAGAATTGGCGAT - 3’(서열번호 34)Forward primer: 5'-GCCACGAATTCCGTAGAATTGGCGAT-3 '(SEQ ID NO: 34)

역방향 프라이머 : 5’-GCCACCTCGAGCGAATCAGAATAATC- 3’(서열번호 35)Reverse primer: 5'-GCCACCTCGAGCGAATCAGAATAATC-3 '(SEQ ID NO: 35)

앞서 설명한 바와 같이, Cag26 단백 유전자를 400 bp씩 중복으로 나누어 17개 절편을 상기 프라이머를 이용하여 중합효소연쇄반응으로 증폭한 후, 유전자재조합기법을 이용하여 대장균의 헬리코박터 파이로리 단백질 발현용 벡터인 pEGexs에 클로닝 하여 발현단백질을 생산하였다. 시발체에는 5' 말단에는 EcoRI 자리와 3' 말단에는 XhoI 자리를 삽입하였다. PCR 증폭 후에는 바로 pGEM-T 벡터에 클로닝 하여 클론을 얻었다. pGEM-T 벡터에 삽입된 유전자 DNA를 EcoRI과 XhoI으로 절단하여 1% 아가로즈젤에 전기영동 하여 분리하고 절편 DNA를 아가로스 겔로부터 분리하여 정제한 후, 상기와 동일한 제한효소를 처리한 pEGexs 벡터를 상기 정제한 DNA와 클 로닝 하였다. As described above, by dividing the Cag26 protein gene by 400 bp in duplicate, 17 fragments were amplified by polymerase chain reaction using the primers, and then, pEGexs, which is a vector for expressing the Helicobacter pylori protein of Escherichia coli, was expressed using a gene recombination technique. Cloning produced the expression protein. In the primer, Eco RI site was inserted at the 5 'end and Xho I site was inserted at the 3' end. After PCR amplification, clones were immediately cloned into pGEM-T vectors to obtain clones. Gene DNA inserted into the pGEM-T vector was digested with Eco RI and Xho I and subjected to electrophoresis on 1% agarose gel. The fragment DNA was isolated from agarose gel, purified, and treated with the same restriction enzyme as above. pEGexs vector was cloned with the purified DNA.

헬리코박터 파이로리의 Cag26 단백의 유전자 절편이 삽입된 pEGexs/Cag26-1, pEGexs/Cag26-2, pEGexs/Cag26-3, pEGexs/Cag26-4, pEGexs/Cag26-5, pEGexs/Cag26-6, pEGexs/Cag26-7, pEGexs/Cag26-8, pEGexs/Cag26-9, pEGexs/Cag26-10, pEGexs/Cag26-11, pEGexs/Cag26-12, pEGexs/Cag26-13, pEGexs/Cag26-14, pEGexs/Cag26-15, pEGexs/Cag26-16 및 pEGexs/Cag26-17을 제조하였으며, 하기 도 1과 같이 도식하였다. PEGexs / Cag26-1, pEGexs / Cag26-2, pEGexs / Cag26-3, pEGexs / Cag26-4, pEGexs / Cag26-5, pEGexs / Cag26-6, pEGexs / Cag26 inserted with gene segments of Cag26 protein of Helicobacter pylori -7, pEGexs / Cag26-8, pEGexs / Cag26-9, pEGexs / Cag26-10, pEGexs / Cag26-11, pEGexs / Cag26-12, pEGexs / Cag26-13, pEGexs / Cag26-14, pEGexs / Cag26-15 , pEGexs / Cag26-16 and pEGexs / Cag26-17 were prepared, as shown in Figure 1 below.

[실시예 2] 헬리코박터 파이로리 Cag26 단백 유전자 절편 단백질 발현Example 2 Helicobacter pylori Cag26 protein fragment protein expression

상기 제조예 1과 같이 제조된 Cag26 단백질의 유전자 절편이 삽입된 클론 단백질의 발현여부를 확인하기 위하여 이를 발현 숙주에 형질전환 시키고 이소프로필티오갈락토시드(isopropyl-β-D-thiogalactoside, IPTG)를 첨가하여 발현을 유도한 결과 모두 효율적으로 발현하였다. In order to confirm the expression of the clone protein into which the gene fragment of the Cag26 protein prepared as in Preparation Example 1 was transformed, it was transformed into an expression host, and isopropyl-β-D-thiogalactoside (IPTG) was used. Expression was induced by addition, and all of them were efficiently expressed.

헬리코박터 파이로리 Cag26 단백질의 유전자 절편이 삽입된 클론은 대장균 DH10B/r에 형질 전환시켜 클로닝된 플라스미드 클론을 얻고 이를 다시 발현 숙주인 대장균 BL21에 형질전환 시켜 이소프로필티오갈락토시드를 처리하여 삽입된 유전자 절편을 발현시켰다. 단백질의 발현여부는 SDS-PAGE(Sodium dodecyl sulfate-polyacryamide gel electrophoresis)를 통하여 확인하였다. The clone into which the gene fragment of Helicobacter pylori Cag26 protein was inserted was transformed into E. coli DH10B / r to obtain a cloned plasmid clone, which was then transformed into E. coli BL21, an expression host, and treated with isopropylthiogalactosid. Was expressed. Protein expression was confirmed by SDS-PAGE (Sodium dodecyl sulfate-polyacryamide gel electrophoresis).

[실시예 3] 헬리코박터 파이로리 Cag26 단백 유전자 절편의 발현단백질의 수용성 분석 Example 3 Solubility Analysis of Expression Proteins of Helicobacter Pylori Cag26 Protein Segment

헬리코박터 파이로리 Cag26 단백의 유전자 절편 pEGexs/Cag26-1, pEGexs/Cag26-2, pEGexs/Cag26-3, pEGexs/Cag26-4, pEGexs/Cag26-5, pEGexs/Cag26-6, pEGexs/Cag26-7, pEGexs/Cag26-8, pEGexs/Cag26-9, pEGexs/Cag26-10, pEGexs/Cag26-11, pEGexs/Cag26-12, pEGexs/Cag26-13, pEGexs/Cag26-15, pEGexs/Cag26-16, pEGexs/Cag26-17를 발현한 후 균체를 파쇄하고 원침하여 상청액과 침전물을 대상으로 SDS-PAGE를 실시한 결과 하기 도 2A와 2B에 나타나 있듯이 pEGexs/Cag26-6, 15, 17의 발현단백질은 수용성으로 발현되었으나 나머지는 불용성으로 발현되었다. 수용성으로 발현되는 양을 하기 도 2B의 화살표 위치에서 비교하면, 수용성으로 발현되는 클론 중 pEGexs/Cag26-6만이 수용성으로 발현되는 양이 절대적으로 많았고 나머지 2개의 클론인 pEGexs/Cag2615 및 pEGexs/Cag26-17은 불용성의 양이 상대적으로 많이 발현되었다. Gene fragments of Helicobacter pylori Cag26 protein pEGexs / Cag26-1, pEGexs / Cag26-2, pEGexs / Cag26-3, pEGexs / Cag26-4, pEGexs / Cag26-5, pEGexs / Cag26-6, pEGexs / Cag26-7, pEGexs / Cag26-8, pEGexs / Cag26-9, pEGexs / Cag26-10, pEGexs / Cag26-11, pEGexs / Cag26-12, pEGexs / Cag26-13, pEGexs / Cag26-15, pEGexs / Cag26-16, pEGexs / Cag26 After expressing -17, the cells were crushed and centrifuged to perform SDS-PAGE on the supernatant and sediment. As shown in FIGS. 2A and 2B, the expression proteins of pEGexs / Cag26-6, 15, and 17 were soluble, but the remainder were expressed. Was expressed insoluble. Comparing the amount expressed in water solubility at the arrow position in FIG. 2B, only pEGexs / Cag26-6 was expressed in water-soluble clones and the remaining two clones, pEGexs / Cag2615 and pEGexs / Cag26- 17 expressed a relatively high amount of insoluble.

[실시예 4] 면역블롯 분석법에 의한 Cag26 단백 유전자 절편 단백질의 항원성 분석[Example 4] Antigen analysis of Cag26 protein fragment protein by immunoblot analysis

본 실시예에서는 Cag26 단백질의 유전자절편이 삽입된 클론의 발현 단백질을 환자혈청을 이용하여 면역블롯 분석법으로 항원성을 분석하였다. Cag26 단백질의 유전자절편이 삽입된 클론의 발현 단백질의 SDS-PAGE 결과를 하기 도 3A에 나타내었으며, 항원의 분석결과는 하기 도 3B와 같다. 그 결과를 요약하면 하기 도 5와 같이 pEGexs/Cag26-1, pEGexs/Cag26-6, pEGexs/Cag26-7, pEGexs/Cag26-10, pEGexs/Cag26-15, 그리고 pEGexs/Cag26-16은 매우 강하게 반응하였고, pEGexs/Cag26-2, pEGexs/Cag26-3, pEGexs/Cag26-8, pEGexs/Cag26-9, pEGexs/Cag26-12, pEGexs/Cag26-13, pEGexs/Cag26-17은 중등도로 반응하였고, pEGexs/Cag26-4, pEGexs/Cag26-5, pEGexs/Cag26-11 및 pEGexs/Cag26-14는 미약한 반응성을 나타내었다. In this example, the antigenicity of the cloned protein-expressing clone of the Cag26 protein was analyzed by immunoblot using patient serum. SDS-PAGE results of the expression protein of the clone into which the gene fragment of Cag26 protein was inserted are shown in FIG. 3A, and the analysis result of the antigen is shown in FIG. 3B. In summary, pEGexs / Cag26-1, pEGexs / Cag26-6, pEGexs / Cag26-7, pEGexs / Cag26-10, pEGexs / Cag26-15, and pEGexs / Cag26-16 react very strongly as shown in FIG. PEGexs / Cag26-2, pEGexs / Cag26-3, pEGexs / Cag26-8, pEGexs / Cag26-9, pEGexs / Cag26-12, pEGexs / Cag26-13, pEGexs / Cag26-17 responded moderately, pEGexs / Cag26-4, pEGexs / Cag26-5, pEGexs / Cag26-11 and pEGexs / Cag26-14 showed weak reactivity.

상기와 같은 결과를 통해 Cag26 단백의 경우 전체적으로 두 군데에서 강한 반응성을 나타내는 도메인이 존재한다는 것을 확인하였다. 즉 N' 말단에 198 아미노잔기 크기의 항원도메인이 있고 염기서열 1999 ~ 2982까지 327 아미노잔기 크기의 항원도메인이 확인되었다. Through the above results, it was confirmed that there are domains showing strong reactivity in two places in the case of Cag26 protein. In other words, an antigen domain of 198 amino residue size was found at the N 'end, and an antigen domain of 327 amino residue size was identified from nucleotide sequence 1999 to 2982.

[제조예 2] 헬리코박터 파이로리의 Cag26-6의 하위 클론제작 Preparation Example 2 Subcloning of Cag26-6 from Helicobacter Pylori

Cag26-6 항원도메인을 구성하는 항원결정기를 확인하기 위하여 Cag26-6에 해당되는 염기서열번호 1999에서 2397 사이를 함유하는 이 Cag26-6 절편보다 짧은 클론 2개를 아래의 프라이머를 이용하여 제작하였다. To identify the epitope constituting the Cag26-6 antigen domain, two clones shorter than this Cag26-6 fragment containing the nucleotide sequences 1999 to 2397 corresponding to Cag26-6 were prepared using the following primers.

Cag26-18Cag26-18

순방향 프라이머 : 5’-GCCACGAATTCATCAGGAGCGAATTG- 3’(서열번호 36)Forward primer: 5'-GCCACGAATTCATCAGGAGCGAATTG- 3 '(SEQ ID NO: 36)

역방향 프라이머 : 5’-GCCACCTCGAGTGCATTAAGGTTTTC- 3’(서열번호 37)Reverse primer: 5'-GCCACCTCGAGTGCATTAAGGTTTTC-3 '(SEQ ID NO: 37)

Cag26-19Cag26-19

순방향 프라이머 : 5’-GCCCCGAATTCGCTTTGAATGATTTC- 3’(서열번호 38)Forward primer: 5'-GCCCCGAATTCGCTTTGAATGATTTC-3 '(SEQ ID NO: 38)

역방향 프라이머 : 5’-GCATACTCGAGTTGCTCTACCTTACT- 3’(서열번호 39)Reverse primer: 5'-GCATACTCGAGTTGCTCTACCTTACT-3 '(SEQ ID NO: 39)

Cag26-18은 염기서열 2050에서 2247번까지이고 Cag26-19는 2248에서 2430까지이다. 제조예 1과 같은 방법으로 pEGexs/Cag26-18와 pEGexs/Cag26-19를 제작하였다. Cag26-18 is from nucleotides 2050 to 2247 and Cag26-19 is from 2248 to 2430. PEGexs / Cag26-18 and pEGexs / Cag26-19 were prepared in the same manner as in Preparation Example 1.

[실시예 5] 헬리코박터 파이로리의 pEGexs/Cag26-18, -19 발현단백질 SDS-PAGE 분석과 면역블롯 분석Example 5 pEGexs / Cag26-18, -19 Expression Protein SDS-PAGE Analysis and Immunoblot Analysis of Helicobacter Pylori

상기 제조예 2에서 제조한 pEGexs/Cag26-18과 19를 대장균에 형질전환하여 발현을 유도하였다. 세포파쇄액 상청액을 니켈컬럼으로 정제하여 SDS-PAGE하고 이를 환자혈청으로 면역블롯을 실시한 결과 하기 도 4와 같다. Cag26-6, Cag26-18 및 Cag26-19를 비교한 결과 Cag26-6의 항체반응성에 비하여 Cag26-18과 Cag26-19의 항원반응성이 급격히 저하되었다. 이러한 결과는 Cag26-6의 항체반응도는 특정 항원결정인자의 반응도에 의존한다기 보다 여러개의 항원결정기 반응도의 합이라고 사료된다. 따라서 Cag26-6은 항원도메인 상태로 발현시키는 것이 혈청학적 진단에 유리할 것으로 판단된다. PEGexs / Cag26-18 and 19 prepared in Preparation Example 2 were transformed into E. coli to induce expression. The cell lysate supernatant was purified by nickel column and subjected to SDS-PAGE, and immunoblot was performed on the patient serum as shown in FIG. 4. Comparing Cag26-6, Cag26-18 and Cag26-19, the antigenic reactivity of Cag26-18 and Cag26-19 was sharply reduced compared to the antibody reactivity of Cag26-6. These results suggest that the antibody reactivity of Cag26-6 is the sum of several epitope reactivity, rather than depending on the specific epitope reactivity. Therefore, expression of Cag26-6 in the antigen domain state may be advantageous for serological diagnosis.

[실시예 6] 헬리코박터 파이로리의 Cag26-6 항원도메인재조합 단백질 정제Example 6 Cag26-6 Antigen Domain Recombination Protein Purification of Helicobacter Pylori

상기 실시예 4와 5의 결과에 따라 수용성으로 발현된 pEGexs/Cag26-6을 선정하였다. pEGexs/Cag26-6의 대장균 클론을 이용하여 상기 실시예 2와 같이 발현시키고 세균체를 수집한 후, 파쇄 원침하여 세포파쇄추출액을 얻었다. 이를 니켈친화성 수지에 적용하여 정제를 시도하였다. 그 결과는 하기 도 6과 같이 이미다졸 200과 300 mM에서 융합단백질이 정제되었다. According to the results of Examples 4 and 5, pEGexs / Cag26-6 expressed in water-soluble form were selected. E. coli clones of pEGexs / Cag26-6 were expressed in the same manner as in Example 2, and the bacterial bodies were collected, followed by crushing to obtain cell disruption extracts. This was applied to a nickel affinity resin to attempt purification. As a result, the fusion protein was purified from imidazole 200 and 300 mM as shown in FIG. 6.

[실시예 7] 헬리코박터 파이로리의 Cag26-6 항원도메인 서열과 융합단백질 서열 분석[Example 7] Cag26-6 antigen domain sequence and fusion protein sequence analysis of Helicobacter pylori

상기 실시예 4에 의해 수용성 Cag26-6 단백의 항원도메인 융합단백질에는 강한 항원도메인 부분인 다음과 같은 서열이 융합되어 있는 것을 확인하였다.In Example 4, it was confirmed that the following sequence, which is a strong antigen domain portion, is fused to the antigen domain fusion protein of the water-soluble Cag26-6 protein.

(서열번호 40)(SEQ ID NO 40)

헬리코박터 파이로리 GroEL 부분인 발현유도단백이 포함된 클론이 발현하는 융합된 Cag26 항원도메인 재조합 단백질 GroELT1/Cag26-6의 아미노산 서열은 다음과 같다.The amino acid sequence of the fused Cag26 antigen domain recombinant protein GroELT1 / Cag26-6, expressed by a clone containing an expression-inducing protein that is a part of Helicobacter pylori GroEL, is as follows.

도 1은 헬리코박터 파이로리의 Cag26 단백 유전자 절편을 도식한 것이다. 1 depicts the Cag26 protein gene fragment of Helicobacter pylori.

도 2는 Cag26 단백 유전자의 불용성 절편클론(A)과 수용성 절편클론인 Cag26-6(B)의 SDD-PAGE의 결과를 나타낸 것이다.Figure 2 shows the results of SDD-PAGE of the insoluble fragment clone (A) of the Cag26 protein gene and Cag26-6 (B) as a water soluble fragment clone.

M; 표준 분자량 마커(Marker)M; Standard Molecular Weight Marker

A 1; pEGexs/Cag26-5의 세포파쇄액 상청액A 1; Cellular lysate supernatant of pEGexs / Cag26-5

A 2; pEGexs/Cag26-5의 세포파쇄액 원침전물A 2; pEGexs / Cag26-5, cell lysate centrifuge

A 3; pEGexs/Cag26-2의 세포파쇄액 상청액A 3; Cellular lysate supernatant of pEGexs / Cag26-2

A 4; pEGexs/Cag26-2의 세포파쇄액 원침전물A 4; pEGexs / Cag26-2, cell lysate centrifuge

A 5; pEGexs/Cag26-4의 세포파쇄액 상청액A 5; Cellular lysate supernatant of pEGexs / Cag26-4

A 6; pEGexs/Cag26-4의 세포파쇄액 원침전물A 6; pEGexs / Cag26-4, cell lysate centrifuge

A 7; pEGexs/Cag26-7의 세포파쇄액 상청액A 7; Cellular fluid supernatant of pEGexs / Cag26-7

A 8; pEGexs/Cag26-7의 세포파쇄액 원침전물A 8; pEGexs / Cag26-7 cell lysate centrifuge

A 9; pEGexs/Cag26-8의 세포파쇄액 상청액A 9; Cellular lysate supernatant of pEGexs / Cag26-8

A 10; pEGexs/Cag26-8의 세포파쇄액 원침전물A 10; pEGexs / Cag26-8, cell lysate centrifuge

A 11; pEGexs/Cag26-9의 세포파쇄액 상청액A 11; Cell lysate supernatant of pEGexs / Cag26-9

A 12; pEGexs/Cag26-9의 세포파쇄액 원침전물A 12; pEGexs / Cag26-9, cell lysate centrifuge

A 13; pEGexs/Cag26-13의 세포파쇄액 상청액A 13; Cell lysate supernatant of pEGexs / Cag26-13

A 14; pEGexs/Cag26-13의 세포파쇄액 원침전물A 14; pEGexs / Cag26-13, cell lysate centrifuge

A 15; pEGexs/Cag26-15의 세포파쇄액 상청액A 15; Cellular fluid supernatant of pEGexs / Cag26-15

A 16; pEGexs/Cag26-15의 세포파쇄액 원침전물A 16; pEGexs / Cag26-15, cell lysate centrifuge

A 17; pEGexs/Cag26-17의 세포파쇄액 상청액A 17; Cellular lysate supernatant of pEGexs / Cag26-17

A 18; pEGexs/Cag26-17의 세포파쇄액 원침전물A 18; pEGexs / Cag26-17, cell lysate stock precipitates

A 19; pEGexs/Cag26-16의 세포파쇄액 상청액 및A 19; cell disruption supernatant of pEGexs / Cag26-16 and

A 20; pEGexs/Cag26-16의 세포파쇄액 원침전물A 20; pEGexs / Cag26-16, cell lysate centrifuge

B 1; pEGexs/Cag26-6의 세포파쇄액 B 1; pEGexs / Cag26-6, cell lysate

B 2; pEGexs/Cag26-6의 세포파쇄액 상청액 및B 2; cell disruption supernatant of pEGexs / Cag26-6 and

B 3; pEGexs/Cag26-6의 세포파쇄액 원침전물B 3; pEGexs / Cag26-6, cell lysate centrifuge

도 3은 Cag26 단백 유전자 절편클론의 발현단백질 SDS-PAGE분석(A), 환자혈청을 이용한 면역블롯분석 결과(B)를 나타낸 것이다. Figure 3 shows the expression protein SDS-PAGE analysis of Cag26 protein fragment clone (A), immunoblot analysis using the patient serum (B).

M; 표준 분자량 마커(Marker)M; Standard Molecular Weight Marker

1; pEGexs/Cag26-1 세포파쇄액 원침전물One; pEGexs / Cag26-1 cell lysate original precipitate

2; pEGexs/Cag26-2 세포파쇄액 원침전물2; pEGexs / Cag26-2 cell lysate original precipitate

3; pEGexs/Cag26-3 세포파쇄액 원침전물3; pEGexs / Cag26-3 cell lysate original precipitate

4; pEGexs/Cag26-4 세포파쇄액 원침전물4; pEGexs / Cag26-4 cell lysate original precipitate

5; pEGexs/Cag26-5 세포파쇄액 원침전물5; pEGexs / Cag26-5 cell lysate original precipitate

6; pEGexs/Cag26-6 세포파쇄액 상청액6; pEGexs / Cag26-6 cell lysate supernatant

7; pEGexs/Cag26-7 세포파쇄액 원침전물7; pEGexs / Cag26-7 cell lysate original precipitate

8; pEGexs/Cag26-8 세포파쇄액 원침전물 8; pEGexs / Cag26-8 cell lysate original precipitate

9; pEGexs/Cag26-9 세포파쇄액 원침전물9; pEGexs / Cag26-9 cell lysate stock solution

10; pEGexs/Cag26-10 세포파쇄액 원침전물10; pEGexs / Cag26-10 cell lysate stock solution

11; pEGexs/Cag26-11 세포파쇄액 원침전물11; pEGexs / Cag26-11 cell lysate original precipitate

12; pEGexs/Cag26-12 세포파쇄액 원침전물12; pEGexs / Cag26-12 cell lysate stock solution

13; pEGexs/Cag26-13 세포파쇄액 원침전물13; pEGexs / Cag26-13 cell lysate original precipitate

14; pEGexs/Cag26-15 세포파쇄액 원침전물14; pEGexs / Cag26-15 cell lysate original precipitate

15; pEGexs/Cag26-16 세포파쇄액 원침전물 및15; pEGexs / Cag26-16 cell lysate original precipitate and

16; pEGexs/Cag26-17 세포파쇄액 원침전물 16; pEGexs / Cag26-17 cell lysate original precipitate

도 4는 pEGexs/Cag26-6와 Cag26-6에 해당되는 DNA 절편 pEGexs/Cag26-18과 Cag26-19 발현단백질의 SDS-PAGE(A)와 면역블롯분석(B)을 나타낸 것이다.4 shows SDS-PAGE (A) and immunoblot analysis (B) of pEGexs / Cag26-6 and Cag26-6 DNA fragments corresponding to pEGexs / Cag26-18 and Cag26-19 expressing proteins.

M; 표준 분자량 마커(Marker)M; Standard Molecular Weight Marker

1; pEGexs/Cag26-6One; pEGexs / Cag26-6

2; pEGexs/Cag26-18 및2; pEGexs / Cag26-18 and

3; pEGexs/Cag26-193; pEGexs / Cag26-19

도 5는 Cag26 유전자 절편단백의 항원성을 도식한 것이다.5 shows the antigenicity of Cag26 gene fragment protein.

1; pEGexs/Cag26-1 절편단백의 항원성 One; Antigenicity of pEGexs / Cag26-1 Fragment Protein

2; pEGexs/Cag26-2 절편단백의 항원성 2; Antigenicity of pEGexs / Cag26-2 Fragment Protein

3; pEGexs/Cag26-3 절편단백의 항원성 3; Antigenicity of pEGexs / Cag26-3 Fragment Protein

4; pEGexs/Cag26-4 절편단백의 항원성 4; Antigenicity of pEGexs / Cag26-4 Fragment Protein

5; pEGexs/Cag26-5 세절편단백의 항원성 5; Antigenicity of pEGexs / Cag26-5 Fragment Protein

6; pEGexs/Cag26-6 절편단백의 항원성 6; Antigenicity of pEGexs / Cag26-6 Fragment Protein

7; pEGexs/Cag26-7 절편단백의 항원성 7; Antigenicity of pEGexs / Cag26-7 Fragment Protein

8; pEGexs/Cag26-8 절편단백의 항원성 8; Antigenicity of pEGexs / Cag26-8 Fragment Protein

9; pEGexs/Cag26-9 절편단백의 항원성 9; Antigenicity of pEGexs / Cag26-9 Fragment Protein

10; pEGexs/Cag26-10 절편단백의 항원성 10; Antigenicity of pEGexs / Cag26-10 Fragment Protein

11; pEGexs/Cag26-11 절편단백의 항원성 11; Antigenicity of pEGexs / Cag26-11 Fragment Protein

12; pEGexs/Cag26-12 절편단백의 항원성 12; Antigenicity of pEGexs / Cag26-12 Fragment Protein

13; pEGexs/Cag26-13 절편단백의 항원성 13; Antigenicity of pEGexs / Cag26-13 Fragment Protein

14; pEGexs/Cag26-15 절편단백의 항원성 14; Antigenicity of pEGexs / Cag26-15 Fragment Protein

15; pEGexs/Cag26-16 절편단백의 항원성 및15; Antigenicity of pEGexs / Cag26-16 Fragment Protein and

16; pEGexs/Cag26-17 세절편단백의 항원성 16; Antigenicity of pEGexs / Cag26-17 Fragment Protein

도 6은 니켈(Nickel) 친화성수지를 이용한 Cag26-6 항원도메인재조합단백질을 정제한 SDS-PAGE 분석 결과를 나타낸 것이다.Figure 6 shows the results of SDS-PAGE analysis of purified Cag26-6 antigen domain recombinant protein using a nickel (Nickel) affinity resin.

M; 표준 분자량 마커(Marker)M; Standard Molecular Weight Marker

1; 세포파쇄액의 컬럼 통과액One; Column through solution of cell lysate

2; 40 mM 이미다졸용액으로 용출한 분획2; Fraction eluted with 40 mM imidazole solution

3; 50 mM 이미다졸으로 용출한 분획3; Fraction eluted with 50 mM imidazole

4; 80 mM 이미다졸으로 용출한 분획4; Fraction eluted with 80 mM imidazole

5; 100 mM 이미다졸으로 용출한 분획5; Fraction eluted with 100 mM imidazole

6; 150 mM 이미다졸으로 용출한 분획6; Fraction eluted with 150 mM imidazole

7; 200 mM 이미다졸으로 용출한 분획 및7; Fraction eluted with 200 mM imidazole and

8; 300 mM 이미다졸으로 용출한 분획8; Fraction eluted with 300 mM imidazole

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY <120> Production of the antigenic domain recombinant protein of Helicobacter pylori Cag26 reactive to human antisera and its method <160> 41 <170> KopatentIn 1.71 <210> 1 <211> 3534 <212> DNA <213> Helicobacter pylori <400> 1 atgactaatg aaaccattga tcaaacaaca acaccagatc aaacactaaa ccaaacggat 60 tttgttccgc aacgatttat ccataatctt caagtagctt ttatcaaagt tgataacgct 120 gtcgcttcat ttgatcctga tcaaaaacca atcgttgata agaatgatag ggataacagg 180 caagcttttg agaaaatctc gcagctaagg gaagaatacg ccaataaagc gatcaaaaat 240 cctgccaaaa agaatcagta tttttcagac tttatcaata agagcaatga tttgatcaac 300 aaagacaatc tcattgctgt agattcttcc gtagatagct ttcggaaatt tggggatcag 360 cgttaccaaa tttttacgag ttgggtgtct cttcaaaaag atccgtctaa aatcaacacc 420 caacaaatcc gaaattttat ggaaaatatc atacaacccc ctatctctga tgataaggaa 480 aaagcggagt ttttgaggtc tgccaaacaa tcttttgcag gaattatcat agggaaccaa 540 atccgatcgg atgaaaaatt catgggcgtg tttgatgaat ctttgaaagc aaggcaagaa 600 gcagaaaaaa atgcagagcc tgctggtggg gattggcttg atattttttt atcatttgta 660 ttcaacaaaa aacaatcttc cgatctcaaa gaaacgctca atcaagagcc aaggcctgat 720 tttgaacaaa atttagccac taccacaacc gacatacaag gcttaccgcc tgaagctaga 780 gatttgcttg atgaaagggg taatttttct aaattcactc ttggtgatat ggaaatgttg 840 gatgttgagg gagtcgctga caaggatccc aattacaagt ttaatcaatt attgatccac 900 aataacgctt tatcttctgt gctaatgggg ggtcatagta acatagaacc tgaaaaggtt 960 tcattattgt atggggataa tggtggtcct gaagctaggc atgattggaa cgccaccgtt 1020 ggttataaaa atcaacaagg caacaatgtg gccacactca ttaatgcgca tcttaataac 1080 ggcagcgggt taatcatagc gggtaatgag gatgggatta aaaatcctag cttctatctc 1140 tacaaagaag accaactcac aggcttgaaa caagcaatga gtcaagaaga gatccaaaac 1200 aaagtagatt tcatggaatt tcttgcgcaa aacaacgcta aattagataa cttgagcgag 1260 aaagagaaag aaaaattcca aactgagatt gaaaatttcc aaaaagaccg taaggcttat 1320 ttggacgctc tagggaatga tcgcattgct tttgtttcta aaaaagaccc aaaacattta 1380 gctttggtta ctgagtttgg taatggggaa gtgagctata ccctcaagga ttatgggaaa 1440 aaacaagata aagctttaga tggggagaca aaaaccactc ttcaaggtaa cctaaaatat 1500 gatggcgtga tgtttgtcaa ttattccaat ttcaaataca ccaacgcctc caagagtcct 1560 gataagggta taggcactac gaatggcgtt tcccatttgg aagcaaattt tagcaaagta 1620 gctgtcttta atttgcctaa tttaaataat ctcgctatca ctaattatat aaggcgagat 1680 ttagaagaga aattgtgggc taaaggattg tccctacaag aagctaataa gctcatcaaa 1740 gactttttga acagcaacaa agaaatggtt agaaaagttt caaacttcaa taaagctgta 1800 gctgaagcta aaaacacagg caattatgat ggagtgaaaa aagctcagaa agatcttgaa 1860 aaatctctaa ggaaacgaga gcatttagag aaagaagtag cgaaaaaatt ggagagcaga 1920 aacgacaaca aaaataaaat ggaagcaaaa gctcaagcta acagccaaaa agataagatt 1980 tttgcactta tcaataaaga ggctagtaag gaagcaagag cggccgcttt cgatccgaat 2040 cttaaaggca tcaggagcga attgtctgat aaacttgaaa atatcaacaa gaatttgaaa 2100 gactttggca aatcttttga tgaactcaaa aatggcaaaa ataaggattt cagcaaggca 2160 gaagaaacgc taaaagccct taaagactcg gtgaaagatt taggcatcaa tccagaatgg 2220 atttcaaaaa ttgaaaacct taatgcagct ttgaatgatt tcaaaaatgg caaaaataag 2280 gatttcagta aggtaacaca agcaaaaagc gaccttgaaa attccattaa ggatgtgatc 2340 attaatcaaa agataacgga taaagttgac aatctcaatc aggctgtatc agagactaaa 2400 ttaacaggcg atttcagtaa ggtagagcaa gccctagccg aactcaaaag cttgtcattg 2460 gatcttggaa aaaattctga tctacaaaaa tccgttaaaa atggtgtaaa tggaacccta 2520 gtcggtaatg ggttatctaa aacagaagcc acaacgctca ccaaaaattt ttcggacatc 2580 aggaaagaat tgaacgagaa gttatttgga aattccaata acaataataa tggactcaaa 2640 aacaacacag agcctattta cgctcaagtt aataaaaaga aaacaggaca agcagctagc 2700 cctgaagagt ccatttacgc tcaagttgct aaaaaggtga gtgcaaaaat tgaccaactc 2760 aacgaatctg catcagcaat aaatagaaaa attgaccgga ttaacaaaat tgcatcagca 2820 ggtaaaggag tgggcggttt cagtggagca gggcgatcag ctagccctga acccatttac 2880 gctacaattg attttgatga ggcaaatcaa gcaggcttcc ctttgaggag atacgctcca 2940 gttgatgatc tcagtaaagt agggctttca agggaacaag aattgactcg tagaattggc 3000 gatctcaatc aggcggtatc agaagctaaa ataggtcatt ttgataacct agaacaaaag 3060 atagatgaac tcaaagattc tacgaaaaag aatgctttga agctatgggt tgaaagcgcg 3120 aaacaagtgc ctactggttt gcaagcgaaa ttggacaatt acgctactaa cagccacaca 3180 cgcattaata gcaatgtcca aactggagcg atcaatgaaa aagcgaccgg catgctaacg 3240 caaaaaaacc ctgagtggct caagctcgtg aatgataaga tagttgcaca taatgtggga 3300 agtgctcatt tgtcagagta tgataaaatt ggattcaacc aaaagaatat gaaagattat 3360 tctgattcgt tcaagttttc caccaagttg aacaacgccg taaaagacat taagtctagc 3420 tttgtgcaat ttttaaccaa tacattttct acaggatctt acagcttgat gaaagcaaat 3480 gcggaacatg gagtcaaaaa tactaataca aaaggtggtt tccaaaaatc ttaa 3534 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Cag26-1 forward pfimer <400> 2 gcaccggaat tcatgactaa tgaaacc 27 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Cag26-1 reverse primer <400> 3 gcgtcgctcg agatcttttt gaagaga 27 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Cag26-2 forward primer <400> 4 gcaccggaat tcttcactct tggtgat 27 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Cag26-2 reverse primer <400> 5 gcgtcgctcg agcatgaaat ctacttt 27 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Cag26-3 forward primer <400> 6 gcaccggaat tcgatttcat ggaatttc 28 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Cag26-3 reverse primer <400> 7 gcatagctcg aggctacttt gctaaaa 27 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Cag26-4 forward primer <400> 8 gcaccggaat tcaaagatcc gtctaaa 27 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Cag26-4 reverse primer <400> 9 gcataactcg agatcaccaa gagtgaa 27 <210> 10 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Cag26-5 forward primer <400> 10 gcacaggaat tcactacgaa tggcgtt 27 <210> 11 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-5 reverse primer <400> 11 gcataactcg aggtcgtttc tgctct 26 <210> 12 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Cag26-6 forward primer <400> 12 gcaatggaat tcgaggctag taaggaa 27 <210> 13 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-6 reverse primer <400> 13 gcataactcg agagtctctg atacag 26 <210> 14 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-7 forward primer <400> 14 gccccgaatt caaattaaca ggcgat 26 <210> 15 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-7 reverse primer <400> 15 gccacctcga gaattttgtt aatccg 26 <210> 16 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-8 forward primer <400> 16 gccacgaatt cgcatcagca ggtaaa 26 <210> 17 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-8 reverse primer <400> 17 gccatctcga gtccagtttg gacatt 26 <210> 18 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-9 forward primer <400> 18 gccatgaatt ccaaactgga gcgatc 26 <210> 19 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-9 reverse primer <400> 19 gccatctcga gtttttggaa accacc 26 <210> 20 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Cag26-10 forward primer <400> 20 gcaccggaat tcaggcaagc ttttgag 27 <210> 21 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 21 gccatcctcg agccttgctt tcaaaga 27 <210> 22 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-11 forward primer <400> 22 gccacgaatt caggcaagaa gcagaa 26 <210> 23 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-11 reverse primer <400> 23 gcctactcga gaaccttttc aggttc 26 <210> 24 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-12 forward primer <400> 24 gccccgaatt ctatggggat aatggt 26 <210> 25 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-12 Cag26-12 <400> 25 gccacctcga gaaccaaagc taaatg 26 <210> 26 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-13 forward primer <400> 26 gccacgaatt cgagtttggt aatggg 26 <210> 27 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-13 reverse primer <400> 27 gccccctcga gtgaaacttt tctaac 26 <210> 28 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-14 forward primer <400> 28 gccccgaatt ccaataaagc tgtagc 26 <210> 29 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-14 reverse primer <400> 29 gcatactcga gcaccgagtc tttaag 26 <210> 30 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-15 forward primer <400> 30 gccccgaatt cgtgaaagat ttaggc 26 <210> 31 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-15 reverse primer <400> 31 gccccctcga gcaattcttt cctgat 26 <210> 32 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-16 forward primer <400> 32 gccccgaatt caaagaattg aacgag 26 <210> 33 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-16 reverse primer <400> 33 gcatactcga gttcttgttc ccttga 26 <210> 34 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-17 forward primer <400> 34 gccacgaatt ccgtagaatt ggcgat 26 <210> 35 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-17 reverse primer <400> 35 gccacctcga gcgaatcaga ataatc 26 <210> 36 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-18 forward primer <400> 36 gccacgaatt catcaggagc gaattg 26 <210> 37 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-18 reverse primer <400> 37 gccacctcga gtgcattaag gttttc 26 <210> 38 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-19 forward primer <400> 38 gccccgaatt cgctttgaat gatttc 26 <210> 39 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-19 reverse primer <400> 39 gcatactcga gttgctctac cttact 26 <210> 40 <211> 133 <212> PRT <213> Helicobacter pylori <400> 40 Glu Ala Ser Lys Glu Ala Arg Ala Ala Ala Phe Asp Pro Asn Leu Lys 1 5 10 15 Gly Ile Arg Ser Glu Leu Ser Asp Lys Leu Glu Asn Ile Asn Lys Asn 20 25 30 Leu Lys Asp Phe Gly Lys Ser Phe Asp Glu Leu Lys Asn Gly Lys Asn 35 40 45 Lys Asp Phe Ser Lys Ala Glu Glu Thr Leu Lys Ala Leu Lys Asp Ser 50 55 60 Val Lys Asp Leu Gly Ile Asn Pro Glu Trp Ile Ser Lys Ile Glu Asn 65 70 75 80 Leu Asn Ala Ala Leu Asn Asp Phe Lys Asn Gly Lys Asn Lys Asp Phe 85 90 95 Ser Lys Val Thr Gln Ala Lys Ser Asp Leu Glu Asn Ser Ile Lys Asp 100 105 110 Val Ile Ile Asn Gln Lys Ile Thr Asp Lys Val Asp Asn Leu Asn Gln 115 120 125 Ala Val Ser Glu Thr 130 <210> 41 <211> 390 <212> PRT <213> Helicobacter pylori <400> 41 Met Ala Lys Glu Ile Lys Phe Ser Asp Ser Ala Arg Asn Leu Leu Phe 1 5 10 15 Glu Gly Val Arg Gln Leu His Asp Ala Val Lys Val Thr Met Gly Pro 20 25 30 Arg Gly Arg Asn Val Leu Ile Gln Lys Ser Tyr Gly Ala Pro Ser Ile 35 40 45 Thr Lys Asp Gly Val Ser Val Ala Lys Glu Ile Glu Leu Ser Cys Pro 50 55 60 Val Ala Asn Met Gly Ala Gln Leu Val Lys Glu Val Ala Ser Lys Thr 65 70 75 80 Ala Asp Ala Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu Ala Tyr 85 90 95 Ser Ile Phe Lys Glu Gly Leu Arg Asn Ile Thr Ala Gly Ala Asn Pro 100 105 110 Ile Glu Val Lys Arg Gly Met Asp Lys Ala Ala Glu Ala Ile Ile Asn 115 120 125 Glu Leu Lys Lys Ala Ser Lys Lys Val Gly Gly Lys Glu Glu Ile Thr 130 135 140 Gln Val Ala Thr Ile Ser Ala Asn Ser Asp His Asn Ile Gly Lys Leu 145 150 155 160 Ile Ala Asp Ala Met Glu Lys Val Gly Lys Asp Gly Val Ile Thr Val 165 170 175 Glu Glu Ala Lys Gly Ile Glu Asp Glu Leu Asp Val Val Glu Gly Met 180 185 190 Gln Phe Asp Arg Gly Tyr Leu Ser Pro Tyr Phe Val Thr Asn Ala Glu 195 200 205 Lys Met Thr Ala Gln Leu Asp Asn Ala Tyr Ile Leu Leu Thr Asp Lys 210 215 220 Lys Ile Ser Ser Met Lys Asp Ile Leu Pro Leu Leu Glu Lys Thr Met 225 230 235 240 Lys Glu Gly Lys Pro Leu Leu Ile Ile Ala Glu Asp Ile Glu Gly Glu 245 250 255 Ala Glu Ala Ser Lys Glu Ala Arg Ala Ala Ala Phe Asp Pro Asn Leu 260 265 270 Lys Gly Ile Arg Ser Glu Leu Ser Asp Lys Leu Glu Asn Ile Asn Lys 275 280 285 Asn Leu Lys Asp Phe Gly Lys Ser Phe Asp Glu Leu Lys Asn Gly Lys 290 295 300 Asn Lys Asp Phe Ser Lys Ala Glu Glu Thr Leu Lys Ala Leu Lys Asp 305 310 315 320 Ser Val Lys Asp Leu Gly Ile Asn Pro Glu Trp Ile Ser Lys Ile Glu 325 330 335 Asn Leu Asn Ala Ala Leu Asn Asp Phe Lys Asn Gly Lys Asn Lys Asp 340 345 350 Phe Ser Lys Val Thr Gln Ala Lys Ser Asp Leu Glu Asn Ser Ile Lys 355 360 365 Asp Val Ile Ile Asn Gln Lys Ile Thr Asp Lys Val Asp Asn Leu Asn 370 375 380 Gln Ala Val Ser Glu Thr 385 390 <110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY <120> Production of the antigenic domain recombinant protein of Helicobacter pylori Cag26 reactive to human antisera and its method <160> 41 <170> KopatentIn 1.71 <210> 1 <211> 3534 <212> DNA <213> Helicobacter pylori <400> 1 atgactaatg aaaccattga tcaaacaaca acaccagatc aaacactaaa ccaaacggat 60 tttgttccgc aacgatttat ccataatctt caagtagctt ttatcaaagt tgataacgct 120 gtcgcttcat ttgatcctga tcaaaaacca atcgttgata agaatgatag ggataacagg 180 caagcttttg agaaaatctc gcagctaagg gaagaatacg ccaataaagc gatcaaaaat 240 cctgccaaaa agaatcagta tttttcagac tttatcaata agagcaatga tttgatcaac 300 aaagacaatc tcattgctgt agattcttcc gtagatagct ttcggaaatt tggggatcag 360 cgttaccaaa tttttacgag ttgggtgtct cttcaaaaag atccgtctaa aatcaacacc 420 caacaaatcc gaaattttat ggaaaatatc atacaacccc ctatctctga tgataaggaa 480 aaagcggagt ttttgaggtc tgccaaacaa tcttttgcag gaattatcat agggaaccaa 540 atccgatcgg atgaaaaatt catgggcgtg tttgatgaat ctttgaaagc aaggcaagaa 600 gcagaaaaaa atgcagagcc tgctggtggg gattggcttg atattttttt atcatttgta 660 ttcaacaaaa aacaatcttc cgatctcaaa gaaacgctca atcaagagcc aaggcctgat 720 tttgaacaaa atttagccac taccacaacc gacatacaag gcttaccgcc tgaagctaga 780 gatttgcttg atgaaagggg taatttttct aaattcactc ttggtgatat ggaaatgttg 840 gatgttgagg gagtcgctga caaggatccc aattacaagt ttaatcaatt attgatccac 900 aataacgctt tatcttctgt gctaatgggg ggtcatagta acatagaacc tgaaaaggtt 960 tcattattgt atggggataa tggtggtcct gaagctaggc atgattggaa cgccaccgtt 1020 ggttataaaa atcaacaagg caacaatgtg gccacactca ttaatgcgca tcttaataac 1080 ggcagcgggt taatcatagc gggtaatgag gatgggatta aaaatcctag cttctatctc 1140 tacaaagaag accaactcac aggcttgaaa caagcaatga gtcaagaaga gatccaaaac 1200 aaagtagatt tcatggaatt tcttgcgcaa aacaacgcta aattagataa cttgagcgag 1260 aaagagaaag aaaaattcca aactgagatt gaaaatttcc aaaaagaccg taaggcttat 1320 ttggacgctc tagggaatga tcgcattgct tttgtttcta aaaaagaccc aaaacattta 1380 gctttggtta ctgagtttgg taatggggaa gtgagctata ccctcaagga ttatgggaaa 1440 aaacaagata aagctttaga tggggagaca aaaaccactc ttcaaggtaa cctaaaatat 1500 gatggcgtga tgtttgtcaa ttattccaat ttcaaataca ccaacgcctc caagagtcct 1560 gataagggta taggcactac gaatggcgtt tcccatttgg aagcaaattt tagcaaagta 1620 gctgtcttta atttgcctaa tttaaataat ctcgctatca ctaattatat aaggcgagat 1680 ttagaagaga aattgtgggc taaaggattg tccctacaag aagctaataa gctcatcaaa 1740 gactttttga acagcaacaa agaaatggtt agaaaagttt caaacttcaa taaagctgta 1800 gctgaagcta aaaacacagg caattatgat ggagtgaaaa aagctcagaa agatcttgaa 1860 aaatctctaa ggaaacgaga gcatttagag aaagaagtag cgaaaaaatt ggagagcaga 1920 aacgacaaca aaaataaaat ggaagcaaaa gctcaagcta acagccaaaa agataagatt 1980 tttgcactta tcaataaaga ggctagtaag gaagcaagag cggccgcttt cgatccgaat 2040 cttaaaggca tcaggagcga attgtctgat aaacttgaaa atatcaacaa gaatttgaaa 2100 gactttggca aatcttttga tgaactcaaa aatggcaaaa ataaggattt cagcaaggca 2160 gaagaaacgc taaaagccct taaagactcg gtgaaagatt taggcatcaa tccagaatgg 2220 atttcaaaaa ttgaaaacct taatgcagct ttgaatgatt tcaaaaatgg caaaaataag 2280 gatttcagta aggtaacaca agcaaaaagc gaccttgaaa attccattaa ggatgtgatc 2340 attaatcaaa agataacgga taaagttgac aatctcaatc aggctgtatc agagactaaa 2400 ttaacaggcg atttcagtaa ggtagagcaa gccctagccg aactcaaaag cttgtcattg 2460 gatcttggaa aaaattctga tctacaaaaa tccgttaaaa atggtgtaaa tggaacccta 2520 gtcggtaatg ggttatctaa aacagaagcc acaacgctca ccaaaaattt ttcggacatc 2580 aggaaagaat tgaacgagaa gttatttgga aattccaata acaataataa tggactcaaa 2640 aacaacacag agcctattta cgctcaagtt aataaaaaga aaacaggaca agcagctagc 2700 cctgaagagt ccatttacgc tcaagttgct aaaaaggtga gtgcaaaaat tgaccaactc 2760 aacgaatctg catcagcaat aaatagaaaa attgaccgga ttaacaaaat tgcatcagca 2820 ggtaaaggag tgggcggttt cagtggagca gggcgatcag ctagccctga acccatttac 2880 gctacaattg attttgatga ggcaaatcaa gcaggcttcc ctttgaggag atacgctcca 2940 gttgatgatc tcagtaaagt agggctttca agggaacaag aattgactcg tagaattggc 3000 gatctcaatc aggcggtatc agaagctaaa ataggtcatt ttgataacct agaacaaaag 3060 atagatgaac tcaaagattc tacgaaaaag aatgctttga agctatgggt tgaaagcgcg 3120 aaacaagtgc ctactggttt gcaagcgaaa ttggacaatt acgctactaa cagccacaca 3180 cgcattaata gcaatgtcca aactggagcg atcaatgaaa aagcgaccgg catgctaacg 3240 caaaaaaacc ctgagtggct caagctcgtg aatgataaga tagttgcaca taatgtggga 3300 agtgctcatt tgtcagagta tgataaaatt ggattcaacc aaaagaatat gaaagattat 3360 tctgattcgt tcaagttttc caccaagttg aacaacgccg taaaagacat taagtctagc 3420 tttgtgcaat ttttaaccaa tacattttct acaggatctt acagcttgat gaaagcaaat 3480 gcggaacatg gagtcaaaaa tactaataca aaaggtggtt tccaaaaatc ttaa 3534 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Cag26-1 forward pfimer <400> 2 gcaccggaat tcatgactaa tgaaacc 27 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Cag26-1 reverse primer <400> 3 gcgtcgctcg agatcttttt gaagaga 27 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Cag26-2 forward primer <400> 4 gcaccggaat tcttcactct tggtgat 27 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Cag26-2 reverse primer <400> 5 gcgtcgctcg agcatgaaat ctacttt 27 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Cag26-3 forward primer <400> 6 gcaccggaat tcgatttcat ggaatttc 28 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Cag26-3 reverse primer <400> 7 gcatagctcg aggctacttt gctaaaa 27 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Cag26-4 forward primer <400> 8 gcaccggaat tcaaagatcc gtctaaa 27 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Cag26-4 reverse primer <400> 9 gcataactcg agatcaccaa gagtgaa 27 <210> 10 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Cag26-5 forward primer <400> 10 gcacaggaat tcactacgaa tggcgtt 27 <210> 11 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-5 reverse primer <400> 11 gcataactcg aggtcgtttc tgctct 26 <210> 12 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Cag26-6 forward primer <400> 12 gcaatggaat tcgaggctag taaggaa 27 <210> 13 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-6 reverse primer <400> 13 gcataactcg agagtctctg atacag 26 <210> 14 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-7 forward primer <400> 14 gccccgaatt caaattaaca ggcgat 26 <210> 15 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-7 reverse primer <400> 15 gccacctcga gaattttgtt aatccg 26 <210> 16 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-8 forward primer <400> 16 gccacgaatt cgcatcagca ggtaaa 26 <210> 17 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-8 reverse primer <400> 17 gccatctcga gtccagtttg gacatt 26 <210> 18 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-9 forward primer <400> 18 gccatgaatt ccaaactgga gcgatc 26 <210> 19 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-9 reverse primer <400> 19 gccatctcga gtttttggaa accacc 26 <210> 20 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Cag26-10 forward primer <400> 20 gcaccggaat tcaggcaagc ttttgag 27 <210> 21 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 21 gccatcctcg agccttgctt tcaaaga 27 <210> 22 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-11 forward primer <400> 22 gccacgaatt caggcaagaa gcagaa 26 <210> 23 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-11 reverse primer <400> 23 gcctactcga gaaccttttc aggttc 26 <210> 24 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-12 forward primer <400> 24 gccccgaatt ctatggggat aatggt 26 <210> 25 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-12 Cag26-12 <400> 25 gccacctcga gaaccaaagc taaatg 26 <210> 26 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-13 forward primer <400> 26 gccacgaatt cgagtttggt aatggg 26 <210> 27 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-13 reverse primer <400> 27 gccccctcga gtgaaacttt tctaac 26 <210> 28 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-14 forward primer <400> 28 gccccgaatt ccaataaagc tgtagc 26 <210> 29 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-14 reverse primer <400> 29 gcatactcga gcaccgagtc tttaag 26 <210> 30 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-15 forward primer <400> 30 gccccgaatt cgtgaaagat ttaggc 26 <210> 31 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-15 reverse primer <400> 31 gccccctcga gcaattcttt cctgat 26 <210> 32 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-16 forward primer <400> 32 gccccgaatt caaagaattg aacgag 26 <210> 33 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-16 reverse primer <400> 33 gcatactcga gttcttgttc ccttga 26 <210> 34 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-17 forward primer <400> 34 gccacgaatt ccgtagaatt ggcgat 26 <210> 35 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-17 reverse primer <400> 35 gccacctcga gcgaatcaga ataatc 26 <210> 36 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-18 forward primer <400> 36 gccacgaatt catcaggagc gaattg 26 <210> 37 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag26-18 reverse primer <400> 37 gccacctcga gtgcattaag gttttc 26 <210> 38 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag 26-19 forward primer <400> 38 gccccgaatt cgctttgaat gatttc 26 <210> 39 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Cag 26-19 reverse primer <400> 39 gcatactcga gttgctctac cttact 26 <210> 40 <211> 133 <212> PRT <213> Helicobacter pylori <400> 40 Glu Ala Ser Lys Glu Ala Arg Ala Ala Ala Phe Asp Pro Asn Leu Lys 1 5 10 15 Gly Ile Arg Ser Glu Leu Ser Asp Lys Leu Glu Asn Ile Asn Lys Asn 20 25 30 Leu Lys Asp Phe Gly Lys Ser Phe Asp Glu Leu Lys Asn Gly Lys Asn 35 40 45 Lys Asp Phe Ser Lys Ala Glu Glu Thr Leu Lys Ala Leu Lys Asp Ser 50 55 60 Val Lys Asp Leu Gly Ile Asn Pro Glu Trp Ile Ser Lys Ile Glu Asn 65 70 75 80 Leu Asn Ala Ala Leu Asn Asp Phe Lys Asn Gly Lys Asn Lys Asp Phe 85 90 95 Ser Lys Val Thr Gln Ala Lys Ser Asp Leu Glu Asn Ser Ile Lys Asp 100 105 110 Val Ile Ile Asn Gln Lys Ile Thr Asp Lys Val Asp Asn Leu Asn Gln 115 120 125 Ala Val Ser Glu Thr 130 <210> 41 <211> 390 <212> PRT <213> Helicobacter pylori <400> 41 Met Ala Lys Glu Ile Lys Phe Ser Asp Ser Ala Arg Asn Leu Leu Phe 1 5 10 15 Glu Gly Val Arg Gln Leu His Asp Ala Val Lys Val Thr Met Gly Pro 20 25 30 Arg Gly Arg Asn Val Leu Ile Gln Lys Ser Tyr Gly Ala Pro Ser Ile 35 40 45 Thr Lys Asp Gly Val Ser Val Ala Lys Glu Ile Glu Leu Ser Cys Pro 50 55 60 Val Ala Asn Met Gly Ala Gln Leu Val Lys Glu Val Ala Ser Lys Thr 65 70 75 80 Ala Asp Ala Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu Ala Tyr 85 90 95 Ser Ile Phe Lys Glu Gly Leu Arg Asn Ile Thr Ala Gly Ala Asn Pro 100 105 110 Ile Glu Val Lys Arg Gly Met Asp Lys Ala Ala Glu Ala Ile Ile Asn 115 120 125 Glu Leu Lys Lys Ala Ser Lys Lys Val Gly Gly Lys Glu Glu Ile Thr 130 135 140 Gln Val Ala Thr Ile Ser Ala Asn Ser Asp His Asn Ile Gly Lys Leu 145 150 155 160 Ile Ala Asp Ala Met Glu Lys Val Gly Lys Asp Gly Val Ile Thr Val 165 170 175 Glu Glu Ala Lys Gly Ile Glu Asp Glu Leu Asp Val Val Glu Gly Met 180 185 190 Gln Phe Asp Arg Gly Tyr Leu Ser Pro Tyr Phe Val Thr Asn Ala Glu 195 200 205 Lys Met Thr Ala Gln Leu Asp Asn Ala Tyr Ile Leu Leu Thr Asp Lys 210 215 220 Lys Ile Ser Ser Met Lys Asp Ile Leu Pro Leu Leu Glu Lys Thr Met 225 230 235 240 Lys Glu Gly Lys Pro Leu Leu Ile Ile Ala Glu Asp Ile Glu Gly Glu 245 250 255 Ala Glu Ala Ser Lys Glu Ala Arg Ala Ala Ala Phe Asp Pro Asn Leu 260 265 270 Lys Gly Ile Arg Ser Glu Leu Ser Asp Lys Leu Glu Asn Ile Asn Lys 275 280 285 Asn Leu Lys Asp Phe Gly Lys Ser Phe Asp Glu Leu Lys Asn Gly Lys 290 295 300 Asn Lys Asp Phe Ser Lys Ala Glu Glu Thr Leu Lys Ala Leu Lys Asp 305 310 315 320 Ser Val Lys Asp Leu Gly Ile Asn Pro Glu Trp Ile Ser Lys Ile Glu 325 330 335 Asn Leu Asn Ala Ala Leu Asn Asp Phe Lys Asn Gly Lys Asn Lys Asp 340 345 350 Phe Ser Lys Val Thr Gln Ala Lys Ser Asp Leu Glu Asn Ser Ile Lys 355 360 365 Asp Val Ile Ile Asn Gln Lys Ile Thr Asp Lys Val Asp Asn Leu Asn 370 375 380 Gln Ala Val Ser Glu Thr 385 390

Claims

서열번호 1의 Cag26 단백 유전자 1999번 내지 2397번 염기서열로 이루어지는 헬리코박터 파이로리(Helicobacter pylori)의 Cag26 단백 항원도메인.Cag26 protein gene of SEQ ID NO: 1 Cag26 protein antigen domain of Helicobacter pylori consisting of the nucleotide sequence of 1999 to 2397.

서열번호 41에 기재된 아미노산 서열로 이루어지는 헬리코박터 파이로리의 Cag26 단백 항원도메인 재조합 단백질.A Cag26 protein antigen domain recombinant protein of Helicobacter pylori consisting of the amino acid sequence set forth in SEQ ID NO: 41.

제 2항에 있어서,3. The method of claim 2,

상기 재조합 단백질은 서열번호 1의 Cag26 단백 유전자 1999번 내지 2397번 염기서열로 이루어지는 헬리코박터 파이로리(Helicobacter pylori)의 Cag26 단백 항원도메인을 포함하는 것을 특징으로 하는 헬리코박터 파이로리 수용성 Cag26 단백 항원도메인 재조합 단백질.The recombinant protein is a Helicobacter pylori water-soluble Cag26 protein antigen domain recombinant protein comprising a Cag26 protein antigen domain of Helicobacter pylori consisting of the Cag26 protein gene of SEQ ID NO: 1999 to 2397.

삭제delete

제 3항에 있어서,The method of claim 3, wherein

상기 항원도메인은 헬리코박터 파이로리 감염환자 항혈청과 반응하는 것을 특징으로 하는 헬리코박터 파이로리 Cag26 단백 항원도메인재조합 단백질.The antigen domain is a Helicobacter pylori Cag26 protein antigen domain recombination protein, characterized in that the reaction with anti-serum of Helicobacter pylori infection.

삭제delete