KR100466382B1 - Recombinant combo protein fused in HIV type-1 and -2, manufacturing method thereof, vector, transformant and diagnostic kit thereof - Google Patents

Recombinant combo protein fused in HIV type-1 and -2, manufacturing method thereof, vector, transformant and diagnostic kit thereof Download PDF

Info

Publication number
KR100466382B1
KR100466382B1 KR10-2002-0000896A KR20020000896A KR100466382B1 KR 100466382 B1 KR100466382 B1 KR 100466382B1 KR 20020000896 A KR20020000896 A KR 20020000896A KR 100466382 B1 KR100466382 B1 KR 100466382B1
Authority
KR
South Korea
Prior art keywords
protein
hiv
gln
leu
combo
Prior art date
Application number
KR10-2002-0000896A
Other languages
Korean (ko)
Other versions
KR20030060274A (en
Inventor
손미진
김은경
유승범
김형철
이상익
오재훈
Original Assignee
주식회사 엘지생명과학
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 주식회사 엘지생명과학 filed Critical 주식회사 엘지생명과학
Priority to KR10-2002-0000896A priority Critical patent/KR100466382B1/en
Publication of KR20030060274A publication Critical patent/KR20030060274A/en
Application granted granted Critical
Publication of KR100466382B1 publication Critical patent/KR100466382B1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus
    • G01N2333/155—Lentiviridae, e.g. visna-maedi virus, equine infectious virus, FIV, SIV
    • G01N2333/16—HIV-1, HIV-2

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Virology (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Zoology (AREA)
  • Biophysics (AREA)
  • Urology & Nephrology (AREA)
  • Physics & Mathematics (AREA)
  • AIDS & HIV (AREA)
  • Cell Biology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Food Science & Technology (AREA)
  • Plant Pathology (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

본 발명은 인간 면역 결핍 바이러스(human immunodeficiency virus; 이하, 'HIV'라 칭한다)-1, -2, O 타입의 단백질이 융합된 유전자 재조합 콤보(Combo) 단백질, 이의 생산방법, 이를 포함하는 벡터 및 형질전환된 대장균, 진단키트 및 백신용 조성물에 관한 것으로, HIV-1, -2, -O형의 각각의 항원성이 높은 단백질을 하나의 단백질(Combo단백질)로 발현되도록 발현벡터를 제조하고, 형질전환된 대장균을 T7/lac 프로모터에 의해 발현 양을 증폭시켰고, 목적단백질, 즉 콤보 단백질의 N-말단에 히스티딘 잔기를 붙여 발현시킴으로 양이온의 히스티딘을 가진 목적단백질을 니켈 음이온이 붙어있는 프로본드(Probond) 칼럼에 흡착시켜 정제하는, 용이한 방법으로 다량의 HIV 콤보 항원을 정제 할 수 있게 제조하는 방법에 관한 것이다The present invention provides a recombinant Combo protein fused to a human immunodeficiency virus (hereinafter referred to as 'HIV')-1, -2, O type protein, a method for producing the same, and a vector comprising the same. The present invention relates to a transformed Escherichia coli, a diagnostic kit, and a composition for a vaccine. An expression vector is prepared to express high antigenic proteins of HIV-1, -2, and -O as one protein (Combo protein), The transformed Escherichia coli was amplified by the T7 / lac promoter, and expressed by attaching a histidine residue to the N-terminus of the target protein, ie, a combo protein, to form a target protein with a cation histidine. Probond) is a method for producing a large amount of HIV combo antigen can be purified by an easy method of adsorption on the column.

Description

인간 면역 결핍 바이러스-1, -2 타입의 단백질이 융합된 재조합 콤보 단백질, 이의 생산방법, 이를 포함하는 벡터, 형질전환된 대장균 및 진단키트{Recombinant combo protein fused in HIV type-1 and -2, manufacturing method thereof, vector, transformant and diagnostic kit thereof}Recombinant combo protein fused in HIV type-1 and -2, manufacturing, recombinant combo protein fused with human immunodeficiency virus-1, -2 type protein, production method thereof, vector comprising the same method according to method, vector, transformant and diagnostic kit

본 발명은 인간 면역 결핍 바이러스(human immunodeficiency virus; 이하, 'HIV'라 칭한다)-1, -2, O 타입의 단백질이 융합된 유전자 재조합 콤보(Combo) 단백질, 이의 생산방법, 이를 포함하는 벡터 및 형질전환된 대장균, 이를 포함하는 진단키트에 관한 것이다.The present invention provides a recombinant Combo protein fused to a human immunodeficiency virus (hereinafter referred to as 'HIV')-1, -2, O type protein, a method for producing the same, and a vector comprising the same. Transformed Escherichia coli, and a diagnostic kit comprising the same.

인간 면역 결핍 바이러스(Human Immunodeficiency Virus; 이하, HIV)는 크게 HIV-1과 HIV-2의 두 가지 형으로 분류되며, HIV-1은 다시 그룹(group) M과 그룹(group) O의 두개의 그룹으로 분류된다. 그룹 M형이 전세계적으로 가장 많이 발견되며 그룹 O형은 주로 중앙 아프리카의 서부지역에 분포한다. 그러나, 이 그룹 O형에 감염된 환자들이 유럽과 미국 등지에서 점차 확인되고 있다. 또한, HIV-2 타입은 서아프리카에서 주로 발견되었으나, 점차적으로 전세계로 확산되고 있다(Charneau P. 등,Virology,205, 247-53, 1994; Gould K. 등,Lancet, 348, 680-1, 1996; Quinn T.C.,Proc Natl Acad Sci USA,91, 2407-14 , 1994).Human Immunodeficiency Virus (HIV) is largely classified into two types, HIV-1 and HIV-2, and HIV-1 is again divided into two groups, group M and group O. Classified as Group M is the most common worldwide, and group O is mainly distributed in western Africa. However, patients infected with this group O are increasingly being identified in Europe and the United States. In addition, HIV-2 type is mainly found in West Africa, but is gradually spreading around the world (Charneau P. et al., Virology, 205, 247-53, 1994; Gould K. et al., Lancet , 348, 680-1, 1996; Quinn TC, Proc Natl Acad Sci USA, 91, 2407-14, 1994).

이들 세 타입은 공통의 항원결정기(epitope)를 가지고 있으나, 상호반응성(cross-reactivity)이 낮아서 HIV-1 타입에 대한 진단시약으로 HIV-2 타입을 검출해내지 못하는 경우가 많으며(Clavel F. 등,Nature, 324, 691-5, 1986; Schable C. 등,Lancet,344, 1333-4, 1994), 최근 국내에도 많은 외국 혈액이 수입됨에 따라, 특히 혈액 스크리닝시 정확한 진단을 위해서는 HIV-1 그룹 M과 HIV-1 그룹 O, HIV-2 타입을 모두 진단할 수 있는 진단시약이 요구되고 있었다.Although these three types have a common epitope, they are often unable to detect HIV-2 type as a diagnostic reagent for HIV-1 type due to low cross-reactivity (Clavel F. et al. , Nature , 324, 691-5, 1986; Schable C. et al., Lancet, 344, 1333-4, 1994), with the recent import of a large number of foreign blood in Korea, especially for the accurate diagnosis of blood screening for the HIV-1 group. There was a need for a diagnostic reagent capable of diagnosing both M, HIV-1 group O, and HIV-2 type.

HIV 감염을 진단하기 위한 표적 중 하나는 바이러스 유전자의 외피유전자(envelope gene;env)을 암호화하는 당단백질 gp41로서(Gnann J.W. Jr. 등,J. Virol., 61, 2639-41, 1987), 항원성이 매우 높으며 이 단백질에 대한 항체는 후천성 면역 결핍증(Acquired Immuno-deficiency Syndrome; 이하 'AIDS'라 칭한다)의 전 과정에 걸쳐서 강하게 지속된다(Weiss R.A.,Science260, 1273-9, 1993). 그러나, 매우 소수성(hydrophobic)인envgp41 단백질의 특성상, 대장균에서env유전자만을 다량으로 발현시키기에 기술적인 어려움이 있었다.One of the targets for diagnosing HIV infection is the glycoprotein gp41, which encodes the envelope gene ( env ) of the viral gene (Gnann JW Jr. et al ., J. Virol ., 61, 2639-41, 1987). Sex is very high and antibodies to this protein persist strongly throughout the entire process of Acquired Immuno-deficiency Syndrome (hereinafter referred to as AIDS) (Weiss RA, Science 260, 1273-9, 1993). However, due to the nature of the very hydrophobic ( env gp41) protein, there was a technical difficulty in expressing only a large amount of the env gene in E. coli.

이러한 문제를 해결하기 위하여 기존의 방법들로는,env유전자와 베타-갈락토시데이즈(β-gal), 말토오즈 결합 단백질(maltose binding protein), GST 유전자 등을 융합하여 용해도 증가와 발현률의 개선의 방법으로 발현이 시도 되었다(Ellinger S. 등,Virology, 180, 811-3, 1991; Morimoto M. 등,AIDS Res. Hum. Retroviruses,9, 971-8, 1993). 그러나, 이러한 방법의 경우에도 이종의 융합단백질이 첨가됨으로 인하여, 정제 후 다시 제거 되어야 하는 문제점이 있었다.In order to solve this problem, existing methods, such as env gene, beta-galactosidase (β-gal), maltose binding protein, GST gene, etc. fused to increase the solubility and improve the expression rate Expression was attempted (Ellinger S. et al., Virology , 180, 811-3, 1991; Morimoto M. et al . , AIDS Res. Hum.Retroviruses, 9, 971-8, 1993). However, even in such a method, since the heterologous fusion protein is added, there has been a problem that it must be removed after purification.

상기와 같은 문제를 해결하기 위하여 본 발명은, 알려진 HIV 타입(type)별 특이성을 갖고 항원성이 뛰어난 융합 단백질을 발현을 위한 발현벡터 및 세포주, 단백질을 제공하는 것을 목적으로 한다.In order to solve the above problems, an object of the present invention is to provide an expression vector, cell line, and protein for expressing a fusion protein having a known specificity for HIV type and excellent antigenicity.

또한, 본 발명은 상기 HIV 타입별 특이성을 갖고 항원성이 뛰어난 융합 단백질을 포함하는 HIV 감염을 진단하는 진단키트 또는 백신제조용 조성물을 제공하는 것을 목적으로 한다.In addition, an object of the present invention is to provide a diagnostic kit or vaccine production composition for diagnosing HIV infection comprising a fusion protein having specificity for each HIV type and excellent antigenicity.

도 1은 콤보(Combo)단백질을 발현하는 플라스미드 pLCSK-1의 제조를 위해 합성한 프라이머를 도시한 것이다(SEQ ID NO:1 내지 6).1 shows primers synthesized for the preparation of plasmid pLCSK-1 expressing the Combo protein (SEQ ID NO: 1 to 6).

도 2는 콤보 단백질의 아미노산 서열을 도시한 것이다(SEQ ID NO:7 내지 11).Figure 2 depicts the amino acid sequence of the combo protein (SEQ ID NOs: 7-11).

도 3은 대장균에서 HIV 콤보 단백질을 발현하는 플라스미드 pLCSK-1의 제조과정을 도시한 공정도이다.Figure 3 is a flow chart showing the manufacturing process of the plasmid pLCSK-1 expressing the HIV combo protein in E. coli.

도 4는 프로본드(Probond) 흡착수지를 이용한 콤보 단백질의 정제된 패턴을 SDS-폴리 아크릴 아마이드 젤 전기영동 결과로 나타낸 사진이다.Figure 4 is a photograph showing the purified pattern of the combo protein using a Probond adsorbent resin as a result of SDS-polyacrylamide gel electrophoresis.

도 5는 HIV -1,-2,O 타입의 양성 환자 혈청으로 웨스턴 블로팅하여 콤보 단백질의 항원성을 검정한 사진이다.5 is a photograph showing Western blot of HIV-1, -2, O type positive patient serum to assay the antigenicity of the combo protein.

도 6은 HIV -1,-2,O 타입의 양성 및 음성 환자 혈청과 정상인의 혈청에서 정제된 콤보 항원을 이용한 효소면역 측정의 특이도 민감도 판정결과를 나타낸 그래프이다.FIG. 6 is a graph showing the results of specificity sensitivity determination of enzyme immunity measurement using purified combo antigens from serum of positive and negative patients of HIV-1, -2, O type and serum of normal persons.

상기와 같은 목적으로 달성하기 위하여 본 발명은, 인간 면역 결핍 바이러스(human immunodeficiency virus, 이하 'HIV'라 칭한다)-1, -2, O 타입의 바이러스들의 항원 중 항원성이 높은것으로, 짜이저(H.L.Zaaijer)등(J. Med. Viro., 51:80-82, 1997) 및 로빈 A. 웨이스(Robin A. Weiss)등(Science, vol. 260, 1273-1278, 1993)에 의해서 알려진 HIV-1 항원단백질 gp41의 일부와 HIV-2 항원 단백질 gp36의 일부, HIV-1의 변이형(variant type)인 O-타입 항원 단백질인 gp41, 그리고 HIV-1 항원단백질인 gag p24 단백질의 유전자를 연결하여 각 바이러스 타입(virus type)의 항원들을 하나의 카이머릭(chimeric) 단백질로 발현을 유도할 수 있는 대장균용 발현벡터 pLCSK-1 및 이를 포함하는 발현세포주 BL21(DE3)/pLCSK-1를 제공한다.In order to achieve the above object, the present invention, human immunodeficiency virus (hereinafter referred to as "HIV")-1, -2, O of the antigen of the virus of type O, the high HLZaaijer et al. (J. Med. Viro., 51: 80-82, 1997) and Robin A. Weiss et al. (Science, vol. 260, 1273-1278, 1993). 1 A portion of the antigenic protein gp41, a portion of the HIV-2 antigen protein gp36, a mutant type of the HIV-1 O-type antigen protein gp41 and the HIV-1 antigen protein gag p24 protein It provides an expression vector pLCSK-1 for E. coli and an expression cell line BL21 (DE3) / pLCSK-1 including the same, which can induce the expression of each virus type antigen as a chimeric protein.

또한 본 발명은 상기 발현벡터가 네 가지의 HIV유래의 융합 단백질을 암호화하는 유전자 서열의 말단에 히스티딘을 암호화하는 코돈을 여섯번 반복되도록 하여, 단백질 발현 유도시, 항원의 용해성 및 발현율 개선의 특성과 함께 C-말단에 히스티딘 잔기가 위치하게 되어, 상용화되어 있는 6×His 태그(tag)을 이용한 단백질 정제방법인 프로본드(Probond, 미합중국 인비트로젠사(Invitrogen Inc.)제품, 카탈로그번호 제R801-01호)를 이용하여 한번의 정제 방법으로 여러 가지의 항원을 동시에 갖는 재조합 HIV 콤보(Combo) 단백질을 다량 제조하는 방법을 제공한다.The present invention also allows the expression vector to repeat the codon encoding histidine six times at the end of the gene sequence encoding the four HIV-derived fusion proteins, with the characteristics of antigen solubility and expression rate improvement when inducing protein expression The histidine residue is located at the C-terminus, and is a protein purification method using a commercially available 6 × His tag, Probond (Invitrogen Inc., US), catalog number R801-01. By using a single purification method) provides a method for producing a large amount of recombinant HIV Combo protein having several antigens at the same time.

보다 상세하게는 본 발명은, HIV 타입(type)-1 p24 또는 gp41, HIV 타입(type)-2 gp36, HIV 타입(type)-1 group O gp41의 전체 혹은 일부를 포함하는 것을 특징으로 하는 단일가닥 폴리펩티드인 융합단백질을 제공한다.More specifically, the present invention includes a single or all of HIV type-1 p24 or gp41, HIV type-2 gp36, and HIV type-1 group O gp41. Provided are fusion proteins that are strand polypeptides.

또한 본 발명은 상기 HIV 타입-1 p24 단백질은 SEQ ID NO:7의 전체 혹은 일부를 포함하고; 상기 HIV 타입-1 gp41 단백질은 SEQ ID NO:8의 전체 혹은 일부를 포함하고; 상기 HIV 타입-2 gp36 단백질은 SEQ ID NO:9의 전체 혹은 일부를 포함하고; 그리고 상기 HIV 타입-1 그룹 O gp41 단백질은 SEQ ID NO:10의 전체 혹은 일부를 포함하는 것을 특징으로 하는 상기 융합단백질을 제공한다.In addition, the present invention the HIV type-1 p24 protein comprises all or part of SEQ ID NO: 7; Said HIV type-1 gp41 protein comprises all or part of SEQ ID NO: 8; Said HIV type-2 gp36 protein comprises all or part of SEQ ID NO: 9; And the HIV type-1 group O gp41 protein provides the fusion protein, which comprises all or part of SEQ ID NO: 10.

또한 본 발명은 여섯번 반복되는 히스티딘 잔기가 상기 융합단백질의 C-말단에 위치하는 것을 특징으로 하는 상기 융합단백질을 제공한다.The present invention also provides the fusion protein, characterized in that six times histidine residues are located at the C-terminus of the fusion protein.

또한 본 발명은 상기 융합단백질을 암호화하는 발현벡터 pLCSK-1를 제공한다.The present invention also provides an expression vector pLCSK-1 encoding the fusion protein.

또한 본 발명은 상기 발현벡터를 포함하는 것을 특징으로 하는 형질전환된 대장균(기탁번호: KCTC 10100BP)을 제공한다.In another aspect, the present invention provides a transformed Escherichia coli (Accession Number: KCTC 10100BP) characterized in that it comprises the expression vector.

또한 본 발명은 배양단계, 균체수집단계, 세포파쇄단계, 분리단계 및 정제단계를 포함하여 이루어지는 융합단백질의 생산방법에 있어서, 제 5항의 상기 형질전환된 대장균을 이용하여 융합단백질을 생산하는 것을 특징으로 하는 융합단백질의 생산방법을 제공한다.In another aspect, the present invention is a method for producing a fusion protein comprising a culture step, cell collection step, cell disruption step, separation step and purification step, characterized in that to produce a fusion protein using the transformed E. coli of claim 5 It provides a method for producing a fusion protein.

또한 본 발명은 상기 정제단계가 융합단백질의 C말단에 있는 6개의 히스티틴 잔기를 프로본드칼럼에 흡착시켜 분리하는 방법에 의하여 이루어짐을 특징으로 하는 상기 융합단백질의 생산방법을 제공한다.In another aspect, the present invention provides a method for producing the fusion protein, characterized in that the purification step is made by adsorbing and separating six histitin residues at the C terminus of the fusion protein to the probond column.

또한 본 발명은 상기의 방법으로 생산된 단백질을 항원으로 사용하는 것을 특징으로 하는 HIV용 진단 키트를 제공한다.In another aspect, the present invention provides a diagnostic kit for HIV, characterized in that using the protein produced by the above method as an antigen.

또한 본 발명은 상기의 방법으로 생산된 단백질을 포함하는 것을 특징으로 하는 백신용 조성물을 제공한다.In another aspect, the present invention provides a vaccine composition comprising a protein produced by the above method.

본 발명에서는 HIV-1 타입의 gp41과 HIV-2 타입의 gp36 중 일부 펩타이드(peptide), HIV-1 그룹(group) O 타입의 gp41을 하나로 연결하여 카이머릭(chimeric) 단백질을 만들고 이의 N-말단에 용해도가 높으며 발현이 잘 되는 HIVgag펩타이드(peptide) p24 단백질을 연결시켜 발현을 유도하므로 이러한 항원의 용해도와 발현율 문제를 개선하였다.(표 1 참조)In the present invention, a peptide of HIV-1 type gp41 and HIV-2 type gp36 and gp41 of HIV-1 group O are linked to form a chimeric protein and its N-terminus. The solubility and expression of HIV gag peptide p24 protein, which is highly soluble, are linked to induce expression, thereby improving the solubility and expression rate of these antigens (see Table 1).

또한, C-말단에는 항원성과 단백질의 발현에 영향을 주지 않는 6개의 히스티딘(histidine)을 붙여서 HIV-1 p24/ HIV-1 gp41/ HIV-2 gp36 펩타이드(peptide)/HIV-1 그룹 O gp41이 하나로 융합된 재조합 단백질을 만들어 대장균에서 발현시킴으로 양이온의 히스티딘을 가진 목적 단백질을 음이온인 니켈이 붙어있는 프로본드(Probond) 칼럼에 흡착시켜 정제하는, 간단한 1단계의(one-step) 방법으로 다량의 HIV 콤보 항원을 정제 할 수 있게 제조하므로 그 정제방법의 개선을 가능하게 하였다.In addition, six histidines that do not affect antigenicity and protein expression are attached to the C-terminus to provide HIV-1 p24 / HIV-1 gp41 / HIV-2 gp36 peptide / HIV-1 group O gp41. By making a recombinant protein fused with E. coli and expressing it in E. coli, a simple one-step method in which a target protein having a cation histidine is adsorbed and purified on a Probond column with nickel anion is attached. Since the HIV combo antigen is manufactured to be purified, the purification method can be improved.

이 결과, 발현량도 상당히 높았으며, HIV-1타입, HIV-2 타입 및 O-타입으로 각각 감염된 AIDS 환자의 혈청으로 웨스턴 블로팅(western blotting)을 시행하여 그 항원성을 조사한 결과, 이 카이머릭 HIV 콤보 항원이 모든 혈액에서 항원성을확보하고 있음을 확인할 수 있었다.As a result, the expression level was also very high, and Western blotting was performed with the serum of AIDS patients infected with HIV-1 type, HIV-2 type and O-type, respectively. It was confirmed that the Merrick HIV combo antigen had antigenicity in all blood.

또한 이종의 단백질을 함유하지 않으므로 종래의 이종의 융합단백질을 함유하는 항원을 사용하는 진단시약에 비하여 보다 높은 특이도를 보유할 수 있다.In addition, since it does not contain heterologous proteins, it may have higher specificity than diagnostic reagents using antigens containing conventional heterologous fusion proteins.

따라서, 본 발명은 향후 보다 높은 특이도와 민감도를 가지는 항원들을 간단한 정제방법으로 다량 확보할 수 있게 하므로 HIV type-1, -2, -O를 진단하는 진단시약 및 백신 등에 유용하게 사용될 수 있다.Therefore, the present invention can secure a large amount of antigens having higher specificity and sensitivity in the future by a simple purification method, so that the present invention can be usefully used for diagnostic reagents and vaccines for diagnosing HIV type-1, -2, and -O.

또한, 이렇게 제조되어진 면역특이적 항원을 유효성분으로서 약제학적으로 허용되는 담체와 혼합하여 예방용 또는 치료용 백신 주사 조성물을 제조할 수 있다.In addition, the immunospecific antigen thus prepared can be mixed with a pharmaceutically acceptable carrier as an active ingredient to prepare a prophylactic or therapeutic vaccine injection composition.

이하 본 발명을 실시예에 의해 구체적으로 기술하면 다음과 같다. 다만 하기 실시예는 본 발명의 예시일 뿐이며 본 발명의 범위를 한정하지는 않는다.Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely illustrative of the present invention and do not limit the scope of the present invention.

<준비단계> HIV의 항원에 대한 유전자의 확보<Preparation stage> Securing genes for HIV antigen

(1) HIV의 항원(HIV-1 p24)에 대한 유전자의 확보(1) Securing the gene for HIV antigen (HIV-1 p24)

HIV-1형 환자의 혈액으로부터 혈장을 취하고 이로부터 그로부터 전체 RNA를 분리하였다. 이때 사용한 시약(reagent)은 TRI 시약(reagent)(미합중국소재 시그마(Sigma)사 제품, 카탈로그 번호 제T9424호)로, 이 용액을 환자의 혈장과 섞은 후 원심분리하여 RNA 층만을 얻은 후, 이를 이소프로파놀(isopropanol)을 이용하여 침전시키고, 에탄올(ethanol)로 씻어낸 후, 이를 다시 3차 증류수에 용해시켰다.Plasma was taken from the blood of an HIV-1 patient and from this the total RNA was separated therefrom. The reagent used at this time is a TRI reagent (Sigma, USA, catalog number T9424). The solution is mixed with the patient's plasma and centrifuged to obtain only an RNA layer. Prepanol (isopropanol) was precipitated, washed with ethanol (ethanol), and then dissolved in tertiary distilled water again.

분리한 RNA로부터 N6 프라이머(대한민국 대전소재 지노테그(Genotech)사 제품)를 이용하여 cDNA를 합성하였다. 이 과정에서는 슈퍼스크립트II(SuperScript II, 미합중국소재 라이프 테크놀로지스(Life Technologies)사 제품, 카탈로그 번호 제18064-022호)라는 역전사효소를 사용하여 cDNA 를 합성하였다.From the isolated RNA, cDNA was synthesized using an N6 primer (Genotech Co., Ltd.). In this process, cDNA was synthesized using a reverse transcriptase called SuperScript II (manufactured by Life Technologies, United States, catalog number 18064-022).

도 1의 프라이머 1과 프라이머 2를 이용하여, 얻어진 cDNA를 주형으로 하여 PCR을 수행하였다. PCR은 94℃, 50℃, 72℃의 순으로 35 순환(cycles)을 수행하였으며, 효소는 벤트 디엔에이 폴리머레이즈(Vent DNA polymerase, 미합중국소재 뉴잉글랜드 바이오랩(New England Biolabs)사 제품, 카탈로그 번호 제254S호)를, PCR 기구(machine)는 미합중국소재 엠제이 리서치(MJ Research)사 제품인 디엔에이 엔진(DNA engine, 모델번호 제PTC-200호)을 이용하였다. 이를 아가로즈 겔(agarose gel)에서 전기 영동을 실시하여 약 700bp의 DNA 조각이 얻어졌음을 확인하였다. 얻어진 DNA 조각을 페놀/클로로포름으로 정제한 후NdeI과BamHI으로 처리하였다.PCR was performed using the obtained cDNA as a template using Primer 1 and Primer 2 of FIG. 1. PCR was performed 35 cycles in the order of 94 ℃, 50 ℃, 72 ℃, the enzyme was Vent DNA polymerase (New England Biolabs, United States, Catalog No. 254S) was used as a DNA machine (DNA engine, model No. PTC-200) manufactured by MJ Research, Inc., USA. Electrophoresis was performed on an agarose gel to confirm that DNA fragments of about 700bp were obtained. The obtained DNA fragment was purified with phenol / chloroform and treated with Nde I and BamH I.

(2) HIV의 항원(HIV-1 gp41', HIV-2 gp36')에 대한 유전자의 확보(2) Securing genes for HIV antigens (HIV-1 gp41 ', HIV-2 gp36')

앞서 HIV-1 p24의 유전자를 얻을 때 사용하였던 cDNA를 주형으로 하고, 도 1의 프라이머 3과 프라이머 4를 이용하여 PCR을 수행하였다. 이 때의 PCR 조건이나 사용한 효소, 기구(machine) 등은 HIV-1 p24 의 유전자를 증폭할 때와 동일하였다. 프라이머 4는 HIV-2 gp36'을 암호화하는 염기서열이 포함되도록 제작하였다. 이를 아가로즈 겔에서 전기 영동하여 약 420bp의 DNA 조각이 얻어졌음을 확인하였다. 얻어진 DNA 조각을 페놀(phenol)/클로로포름(chloroform)으로 정제한 후BamHI과EcoRI으로 처리하였다.CDNA was used as a template for obtaining the gene of HIV-1 p24, and PCR was performed using primers 3 and 4 of FIG. At this time, PCR conditions, enzymes and machines used were the same as those for amplifying the gene of HIV-1 p24. Primer 4 was constructed to include a base sequence encoding HIV-2 gp36 '. It was electrophoresed on an agarose gel to confirm that DNA fragments of about 420 bp were obtained. The obtained DNA fragment was purified with phenol / chloroform and treated with BamH I and EcoR I.

(3) HIV의 항원(HIV-O gp41')에 대한 유전자의 확보(3) Securing the gene for HIV antigen (HIV-O gp41 ')

HIV-O의 환자 혈액으로부터 혈장을 취하고 이로부터 전체 RNA를 분리하였다. 이 때에도, HIV-1 p24 의 유전자를 얻을 때와 마찬가지로 TRI 시약(reagent), 이소프로파놀(isopropanol), 에탄올(ethanol) 등을 사용하였다. 분리한 RNA로부터 N6 프라이머(대한민국 대전 소재, 지노테크(Genotech)사 제품)를 이용하여 cDNA를 합성하였다.Plasma was taken from patient blood of HIV-O and total RNA was isolated therefrom. At this time, TRI reagent, isopropanol, ethanol and the like were used as in the case of obtaining the gene of HIV-1 p24. From the isolated RNA, cDNA was synthesized using an N6 primer (Genotech, Daejeon, Korea).

도 1의 프라이머 5과 프라이머 6을 이용하여, 얻어진 cDNA를 주형으로 하는 PCR을 수행하였다. 이를 아가로즈 겔에서 전기영동을 실시하여 약 320bp의 DNA 조각이 얻어졌음을 확인하였다. 얻어진 DNA 조각을 페놀(phenol)/ 클로로포름(chloroform)으로 정제한 후EcoRI과XhoI으로 처리하였다.Using primers 5 and 6 of Fig. 1, PCR was performed using the obtained cDNA as a template. Electrophoresis was performed on an agarose gel to confirm that DNA fragments of about 320bp were obtained. The obtained DNA fragment was purified with phenol / chloroform and treated with EcoR I and Xho I.

<실시예 1> 재조합 발현벡터 pLCSK-1의 제작 및 균주의 형질전환Example 1 Preparation of recombinant expression vector pLCSK-1 and transformation of strain

pET23a(미합중국 소재 노바젠(Novagen)사 제품, 카탈로그 번호 제69745-3호)를NdeI과XhoI으로 처리하고, 이를 상기 과정에서 얻어진 세가지 종류의 DNA 조각과 함께 라이게이션(ligation) 반응을 수행하였다. 이 때 사용한 효소는 T4 DNA 라이게이즈(ligase)(미합중국 소재 로슈(Roche)사 제품, 카탈로그 번호 제716 359호)였으며, 16℃에서 하루동안(overnight) 반응시킨 후, 형질전환(transformation) 과정을 수행하였다. 이를 통하여 얻어진 형질전환체로부터 약 5.1kb크기의 발현벡터를 확인할 수 있었고, 이를 pLCSK-1이라 하였다(도 3).pET23a (Novagen, USA, catalog number 69745-3) was treated with Nde I and Xho I and subjected to ligation reaction with the three kinds of DNA fragments obtained in the above process. It was. The enzyme used at this time was a T4 DNA ligase (Roche, USA, catalog No. 716 359), which was transformed after an overnight reaction at 16 ° C. Was performed. An expression vector having a size of about 5.1 kb was confirmed from the obtained transformant, which was called pLCSK-1 (FIG. 3).

이렇게 얻어진 발현벡터를 대장균 BL21(DE3)(Studier FW 및 Moffatt BA,J. Mol. Bio., 189(1), 113-30, 1986)에 형질전환시켰다.The expression vector thus obtained was transformed into Escherichia coli BL21 (DE3) (Studier FW and Moffatt BA, J. Mol. Bio ., 189 (1), 113-30, 1986).

형질전환체를 암피실린(50mg/ml)이 첨가된 LB배지에서 하루밤 배양하였고,이에서 선별된 재조합 발현균주 BL21(DE3)/pLCSK-1를 생명공학 연구원내 유전자 은행에 균주로 기탁하였다(기탁번호 KCTC10100BP).The transformants were incubated overnight in LB medium supplemented with ampicillin (50 mg / ml), and the recombinant expression strain BL21 (DE3) / pLCSK-1 selected therefrom was deposited as a strain in the gene bank of the Biotechnology Research Institute (Accession No. KCTC10100BP).

<실시예 2> 재조합 균주에서의 HIV 콤보 단백질의 발현 및 정제Example 2 Expression and Purification of HIV Combo Proteins in Recombinant Strains

상기 기탁된 재조합 발현 균주 BL21(DE3).pLCSK-1(기탁번호 KCTC10100BP)를 암피실린(50mg/ml)이 첨가된 LB 배지 3ml에 접종하고, 37℃에서 진탕배양하여 600 nm에서의 흡광도가 0.8이 되도록 하고(스펙트로포토미터(spectrophotometer)는 노바스펙II(Novaspec II visible, 미합중국소재 아머시암-파마시아(Amersham-Pharmacia)사 제품, 카탈로그 번호 제80-2088-64호)를 사용하였다), 600nm에서의 흡광도가 0.8에 이르렀을 때, 최종농도가 1mM이 되도록 IPTG(Isopropylthiogalactoside)를 첨가하고, 4시간을 더 배양하였다.The deposited recombinant expression strain BL21 (DE3) .pLCSK-1 (Accession No. KCTC10100BP) was inoculated in 3 ml of LB medium to which ampicillin (50 mg / ml) was added, and shaken at 37 ° C. to obtain an absorbance of 0.8 nm at 600 nm. (Spectrophotometer was used Novaspec II visible (Amersham-Pharmacia, US Catalog No. 80-2088-64) at 600nm) When the absorbance reached 0.8, IPTG (Isopropylthiogalactoside) was added so as to have a final concentration of 1 mM, and further cultured for 4 hours.

배양액의 1ml을 원심분리하여 균체만을 회수한 다음, PBS(Phosphate-buffered saline), pH 7.4, 100㎕를 첨가하여 재분산시킨 후, SDS-샘플 버퍼(sample buffer) 50㎕를 첨가하여 100℃에서 5분 동안 열을 가해 주었다. 이를 14,000rpm에서 10분동안 원심분리하여 상층액만을 회수하였다. 이후, 12% SDS-PAGE(Laemmli UK,Nature227, 680-5, 1970)를 행하여 분자량 50kDa위치의 단백질의 발현이 강하게 나타나는 것을 확인하였다(도 4).Centrifugation of 1ml of the culture solution to recover only the cells, and then redispersed by adding PBS (Phosphate-buffered saline), pH 7.4, 100μl, 50μl of SDS-sample buffer (additional) at 100 ℃ Heated for 5 minutes. This was centrifuged at 14,000 rpm for 10 minutes to recover only the supernatant. Thereafter, 12% SDS-PAGE (Laemmli UK, Nature 227, 680-5, 1970) was performed to confirm that the expression of the protein at the molecular weight of 50 kDa was strongly expressed (FIG. 4).

단백질 정제를 위해 재조합 발현 균주 BL21(DE3).pLCSK-1(기탁번호 KCTC 10100 BP)를 암피실린(50mg/ml)이 첨가된 LB 배지 30ml에 접종하고, 37℃에서 18시간 동안 진탕배양한 다음, 포화된 배양액을 동일한 LB 배지 1L에 접종하여 600 nm에서의 흡광도가 0.8이 될 때까지 37℃에서 진탕배양하였다. 600nm에서의 흡광도가 0.8에 이르렀을 때, 최종농도가 1mM이 되도록 IPTG(Isopropylthiogalactoside)를 첨가하고, 4시간을 더 배양하였다. 이 진탕배양액 1L을 원심 분리(6,000rpm, 4℃, 20분, 미합중국소재 베크만 코울터사(Beckman Coulter, Inc.)제품, 제품명 베크만(Beckman) J2-21M 원심분리기(Centrifuge), 2 X 500ml, 카탈로그 번호 제344301호)하여 배양액을 버리고 균체만을 취하여 분산액(PBS, pH 7.4) 30ml에 재분산시켰다. 분산시킨 균체는 초음파 파쇄기(미합중국 브랜슨(Branson)사 제품, 제품명 소니파이어450(Sonifier 450)를 이용하여 파쇄하였다.For protein purification, recombinant expression strain BL21 (DE3) .pLCSK-1 (Accession No. KCTC 10100 BP) was inoculated in 30 ml of LB medium supplemented with ampicillin (50 mg / ml), and shaken at 37 ° C. for 18 hours. Saturated cultures were inoculated in 1 L of the same LB medium and shaken at 37 ° C until the absorbance at 600 nm was 0.8. When the absorbance at 600 nm reached 0.8, IPTG (Isopropylthiogalactoside) was added so as to have a final concentration of 1 mM, and further cultured for 4 hours. Centrifuge 1 L of this shake culture solution (6,000 rpm, 4 ° C., 20 minutes, Beckman Coulter, Inc., USA), Beckman J2-21M Centrifuge, 2 X 500 ml, Catalog No. 344301), the culture medium was discarded, and only the cells were taken and redispersed in 30 ml of dispersion (PBS, pH 7.4). The dispersed cells were crushed using an ultrasonic crusher (Branson, USA, product name Sonyfire 450).

발현된 HIV 콤보 단백질은 소수성 부위를 포함하므로 원심분리한 침전물의 구역에 존재하므로 원심 분리(14,000rpm, 4℃, 30분, 일본국 소재 히타치사(Hitachi Ltd.)제품, 제품명 하이멕(himac) CR 22F, 50ml, 카탈로그 번호 제S101521B호)한 후, 상층액을 버리고 남은 침전물을 6M 구아니딘(guanidine)-HCl로 용해하여 HIV 콤보 단백질을 함유하는 용액을 수집하였다.Since the expressed HIV combo protein contains a hydrophobic site, it exists in the zone of centrifuged sediment, so it is centrifuged (14,000 rpm, 4 ° C., 30 minutes, Hitachi Ltd., Japan, product name himac). CR 22F, 50 ml, Cat. No. S101521B), the supernatant was discarded, and the remaining precipitate was dissolved in 6M guanidine-HCl to collect a solution containing HIV combo protein.

이후, HIV 콤보 단백질의 정제를 위해서, 단백질 발현시 HIV 콤보 단백질의 C-말단에 발현되도록한 6개의 히스티딘 잔기를 이용하여 프로본드(Probond) 칼럼(미합중국 소재 인비트로젠사(Invitrogen Inc.)제품, 카탈로그 번호 제R801-01호)에 흡착시켰다. 흡착 되지않는 불순물은 6M 구아니딘(guanidine)-HCl과 20mM 이미다졸(imidazol)이 들어있는 PBS(pH 7.4)로 세척하여 제거하고, 흡착되어 있던 표면항원은 6M 구아니딘(guanidine)-HCl과 300mM 이미다졸이 들어있는 PBS(pH 7.4)로 탈착시켜 분리하였다.Subsequently, for purification of the HIV combo protein, a Probond column (Invitrogen Inc., USA) using six histidine residues that were expressed at the C-terminus of the HIV combo protein upon protein expression. And catalog number R801-01). Unadsorbed impurities are removed by washing with PBS (pH 7.4) containing 6M guanidine-HCl and 20 mM imidazol, and the adsorbed surface antigens are 6M guanidine-HCl and 300 mM imidazole. It was separated by desorption with PBS (pH 7.4).

이 과정에서 표면항원(SEQ ID NO:10)은 SDS PAGE결과 분자량 50Kd에서 95%이상 순수분리 되었음을 알수 있었다(도 4). 이 분리용액을 8M 요소로 투석하여 진단키트를 포함하는 다양한 용도의 구성물로서 사용하였다.In this process, the surface antigen (SEQ ID NO: 10) was found to be more than 95% pure separation of the molecular weight 50Kd SDS PAGE results (Fig. 4). This separation solution was dialyzed with 8M urea and used as a component for various uses including diagnostic kits.

<실시예 3> 대장균에서 정제된 HIV 콤보 단백질의 항원성 검정Example 3 Antigen Assay of HIV Combo Protein Purified in Escherichia Coli

상기 실시예 2에서 12% SDS-PAGE를 행하여 분자량 50Kd위치의 HIV 콤보 단백질의 발현을 확인하였으며, 이 단백질의 항원성을 검정하기 위해 AIDS 환자 혈청으로 웨스턴 블로팅(Western blotting)을 실시하였다.In Example 2, 12% SDS-PAGE was performed to confirm the expression of the HIV combo protein having a molecular weight of 50 Kd, and Western blotting was performed with AIDS patient serum to test the antigenicity of the protein.

HIV 콤보 단백질의 발현이 유도된 세포 파쇄액 또는 정제된 HIV 콤보 단백질 용액을 12% SDS-PAGE에서 전기영동 하고나서 남은 겔을 30분 동안 25mM 트리스(Tris)-HCl, 190mM 글리신(pH 8.3), 20% 메탄올 용액에 담궈 둔 후, 같은 완충용액을 이용하여 니트로셀룰로즈 용지로 단백질을 옮겼다. 단백질이 옮겨진 니트로셀룰로즈 용지를 1% 소혈청알부민(Bovine Serum Albumin, BSA)/PBS, pH 7.4 완충용액으로 상온에서 1시간동안 블로킹(blocking)하고, 1/100으로 희석한 각 AIDS 환자 혈청(HIV-1 타입(type), HIV-2 타입(type), HIV-1 그룹(group) O 타입)으로 상온에서 1시간동안 반응시켰다. 0.05% 트윈/PBS 용액으로 반응이 끝난 용지를 5분 간격으로 5회 세척 후, 1/2000로 희석된 염소에서 만든 과산화효소가 접합된 2차 항체(HRP-conjugated anti-human goat secondary antibody; 미합중국소재 백터랩스(Vector Labs)사 제품)를 상온에서 1시간동안 반응시켰다. 이후 니트로셀룰로즈 용지를 0.05% 트윈/PBS 용액으로 세척한 후 4-클로로-나프톨(4-chloro-naphthol)과 과산화수소로 발색을 유도하였다(Towbin 등,Proc. Natl. Acad. Sci. USA,76, 4350-4, 1979).After the cell lysate induced by the expression of the HIV combo protein or the purified HIV combo protein solution was electrophoresed in 12% SDS-PAGE, the remaining gel was immersed in 25 mM Tris-HCl, 190 mM glycine (pH 8.3) for 30 minutes, After soaking in 20% methanol solution, the protein was transferred to nitrocellulose paper using the same buffer solution. Protein-transferred nitrocellulose paper was blocked with 1% Bovine Serum Albumin (BSA) / PBS, pH 7.4 buffer for 1 hour at room temperature, and diluted to 1/100 of each AIDS patient serum (HIV). -1 type, HIV-2 type, and HIV-1 group O type) were reacted at room temperature for 1 hour. Wash the finished paper with 0.05% Tween / PBS solution 5 times at 5 minute intervals, and then use HRP-conjugated anti-human goat secondary antibody conjugated with 1/2000 diluted goat. Vector Labs, Inc.) was reacted at room temperature for 1 hour. The nitrocellulose paper was then washed with 0.05% Tween / PBS solution and then developed with 4-chloro-naphthol and hydrogen peroxide (Towbin et al., Proc. Natl. Acad. Sci. USA, 76, 4350-4, 1979).

그 결과 세가지 서로 다른 HIV-아종(subtype)에 대한 혈청에 의해서 HIV 콤보 단백질이 검출됨을 볼 수 있었고, 모두 특이적인 50kDa의 밴드만을 검출하는 것으로 보아 HIV 콤보 단백질의 항원특이성을 검정 할 수 있었다(도 5).As a result, HIV combo protein was detected by sera for three different HIV-subtypes, and all of them detected only a specific band of 50kDa, and thus the antigen specificity of HIV combo protein could be tested (Fig. 5).

<실시예 4> HIV 항체 측정(assay)용 96-plate 제작Example 4 Preparation of 96-plate for HIV Antibody Assay

효소 면역 측정(Enzyme Immuno Assay, EIA)용 96웰 플레이트(well plate)(미합중국 소재 넌크(Nunc)사 제품)에 실시예 2에서 정제한 콤보 항원 용액을 1㎍/ml의 농도로 0.1M 탄산 염화 완충용액(pH 9.5)에 희석 하여 100㎕씩 분주 하였다. 플레이트(plate)를 밀봉하고 4℃에서 18시간 방치하여 항원이 웰(well)에 부착되게 하였다. 이후 항원용액을 제거 하고 0.1% BSA을 함유한 PBS를 웰(well)당 300㎕씩 분주하고 4℃에서 18시간 방치 하였다. 웰(well)에 있는 용액을 제거 하고 상온에서 1시간 동안 방치하여 수분을 건조시킨 후 제습제와 함께 밀봉용기에 넣어 4℃ 저온 냉장고에 보관하고 필요시 사용하였다.0.1 M carbonate in a 96-well plate (Nunc, U.S.), a combo antigen solution purified in Example 2 for the enzyme immunoassay (Enzyme Immuno Assay, EIA) at a concentration of 1 µg / ml Diluted in buffer solution (pH 9.5) and dispensed 100ul. The plate was sealed and left at 4 ° C. for 18 hours to allow antigen to adhere to the wells. Thereafter, the antigen solution was removed, and 300 μl of PBS containing 0.1% BSA was dispensed per well, and left at 4 ° C. for 18 hours. The solution in the well (well) was removed and left at room temperature for 1 hour to dry the moisture, and then placed in a sealed container with a dehumidifying agent and stored in a 4 ℃ low temperature refrigerator and used if necessary.

<실시예 5> 콤보 항원을 이용한 HIV 항체 간접 효소면역측정(indirect EIA assay)Example 5 Indirect EIA Assay for HIV Antibodies Using Combo Antigen

실시예 2의 방법으로 얻어진 재조합 콤보 항원을 실시예 4와 같은 방법으로 96웰-플레이트(well-plate)에 단백질을 코팅하여 측정(assay)에 사용할 플레이트(plate)를 제조 하고, 제조된 플레이트의 각 웰에 음성 혈장 샘플(sample)과 항체 양성 샘플을 각각 20㎕씩 첨가하고 시료희석용액(PBS buffer)을 100㎕를 첨가하여 잘 혼합한 다음 37℃ 반응기에 넣고 60분간 반응 시켰다. 트윈20(Tween 20)을 함유한 PBS 300㎕씩 5회 웰을 세정 하고 과산화효소(peroxidase)가 결합된이차항체를 넣은 후 30분 동안 37℃에서 반응한 후, 다시 트윈20(Tween 20)을 함유한 PBS 300㎕씩 5회 웰을 세정 하고 기질용액(테트라메틸 벤지딘 100㎍/㎖, 과산화수소 0.006%, 구연산 인산염 완충용액 pH 4.5)을 100㎕씩 첨가하였다. 30분간 암소에서 발색 시킨 후 반응 정지액(2N 황산용액) 100㎕를 각 웰에 첨가하고 96 플레이트 판독기(미합중국 소재 몰레큘러 디바이시즈(Molecular Devices)사 제품)로 650nm를 보조(referance) 파장으로 하고 450nm에서 흡광도를 측정 하였다. 컷오프(Cut-off)를 음성시료의 평균값에 0.2를 더한 값으로 결정하고, 위의 방법으로 50개의 HIV PCR 양성시료와 50개의 음성시료를 실험하였다. 그 결과 50개의 양성시료 중 50개를 양성으로 판정하였고, 음성시료의 경우 50개중 50개를 음성으로 판정하여 100%의 민감도와 100%의 높은 특이도를 보였다(도 6).The recombinant combo antigen obtained by the method of Example 2 was coated with a protein in a 96 well-plate in the same manner as in Example 4 to prepare a plate for use in assays, and the Negative plasma samples (samples) and antibody positive samples were added to each well 20µl, and 100µl of sample dilution solution (PBS buffer) was added and mixed well. The wells were washed five times with 300 μl of PBS containing Tween 20, and the reaction was performed at 37 ° C. for 30 minutes after adding a secondary antibody conjugated with peroxidase, followed by Tween 20. The wells were washed five times with 300 μl of PBS containing 100 μl of substrate solution (tetramethyl benzidine 100 μg / ml, hydrogen peroxide 0.006%, citric acid phosphate buffer pH 4.5). After 30 minutes of color development in the dark, 100 µl of the reaction stopper (2N sulfuric acid solution) was added to each well, and 96 nm reader (Molecular Devices, USA) was used as the reference wavelength of 650 nm. Absorbance was measured at 450 nm. Cut-off was determined by adding 0.2 to the average of negative samples, and 50 HIV PCR positive samples and 50 negative samples were tested by the above method. As a result, 50 out of 50 positive samples were determined to be positive, and 50 out of 50 positive samples were determined to be negative, showing 100% sensitivity and 100% high specificity (FIG. 6).

<실시예 6> 타사 제품과의 민감도 비교 실험Example 6 Sensitivity Comparison Experiment with Other Companies

콤보 항원의 역가, 즉 민감도 특이도를 기존의 재조합 HIV항원과 비교하여 하기의 표 1에 나타내었다. 이들 실험에서 사용한 항원의 양은 대략 1㎍/웰(well)로서 대부분 효소면역 측정법에 의하여 수행되었다.The titer of the combo antigen, ie sensitivity specificity, is shown in Table 1 below in comparison to the existing recombinant HIV antigens. The amount of antigen used in these experiments was approximately 1 μg / well, mostly carried out by enzyme immunoassay.

항원antigen 특이도(%)% Specificity 민감도(%)responsiveness(%) 수율yield 비고Remarks P24gag:100-309P24 gag : 100-309 100100 99.599.5 -- Ellinger 등, Virology, 180, 811(1991)Ellinger et al., Virology, 180, 811 (1991) P121Env:439-640P121 Env : 439-640 99.899.8 99.099.0 4mg/g4mg / g Ghrayeb 등,HIV detection by genetic Engineering Method, p41Ghrayeb et al., HIV detection by genetic Engineering Method, p41 로슈 HIVgag/env Roche HIV gag / env 99.899.8 99.599.5 2.5-3mg/g2.5-3mg / g Shoeman 등,HIV detection by genetic Engineering Method, p99Shoeman et al., HIV detection by genetic Engineering Method, p99 콤보Combo 100100 100100 15-20mg/l15-20mg / l 본 발명The present invention

상기 표 1에서 보는 바와 같이 본 발명의 콤보 항원은 p24 혹은env단백질 단독으로 사용된 경우와gag/env를 각각 발현하여 섞어 사용한 경우보다 특이도와 민감도에서 더욱 우수함을 알 수 있었다.As shown in Table 1, the combo antigen of the present invention was found to be superior in specificity and sensitivity than in the case of using p24 or env protein alone and using a mixture of gag / env .

이상에서 본 바와 같이, 본 발명은 종래의 항원 진단표적에 비하여 높은 발현양과 손쉬운 정제가 가능하고, 항체에 대한 민감도와 특이성이 높아서, HIV 감염에 대한 진단 시약 또는 HIV 백신 제조용 조성물로 사용될 수 있는 유전자 재조합에 의해 발현된 HIV 콤보 단백질 및 이를 포함하는 벡터와 형질전환된 대장균, 그리고 이를 이용한 콤보 단백질의 제조방법을 제공하는 유용한 발명이다.As described above, the present invention is a gene that can be used as a diagnostic reagent or HIV vaccine composition for HIV infection due to high expression and easy purification, high sensitivity and specificity for antibodies compared to conventional antigen diagnostic targets It is a useful invention to provide a recombinantly expressed HIV combo protein, a vector comprising the same and transformed E. coli, and a method for producing the combo protein using the same.

<110> SUNG Jae Gap; LG CHEM INVESTMENT, LTD. <120> Recombinant combo protein fused in HIV type-1, -2 and 0, manufacturing method thereof, vector and transformant thereof, diagnostic kit thereof and composition for vaccine thereof <130> NK-0251 <160> 11 <170> KopatentIn 1.71 <210> 1 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer 1 <400> 1 aaacatatgc agaacctcca ggggcaa 27 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer 2 <400> 2 aaaggatccc aaaactcttg ctttatg 27 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer 3 <400> 3 aaaggatccg gcgcagcgtc aatgacg 27 <210> 4 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> Primer 4 <400> 4 aaagaattcg ttaacagtag tgtggcaaac ctgacggaaa gcgcaacccc aggagttcag 60 acgagcctgg tctaagcttg tgtaattgtt 90 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer 5 <400> 5 aaagaattca tacaggccca gcagcaa 27 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer 6 <400> 6 aaactcgaga ttttgttcct gctgtac 27 <210> 7 <211> 231 <212> PRT <213> Human immunodeficiency virus type 1 p24 <400> 7 Pro Ile Val Gln Asn Leu Gln Gly Gln Met Val His Gln Ala Ile Ser 1 5 10 15 Pro Arg Thr Leu Asn Ala Trp Val Lys Val Val Glu Glu Lys Ala Phe 20 25 30 Ser Pro Glu Val Ile Pro Met Phe Ser Ala Leu Ser Glu Gly Ala Thr 35 40 45 Pro Gln Asp Leu Asn Thr Met Leu Asn Thr Val Gly Gly His Gln Ala 50 55 60 Ala Met Gln Met Leu Lys Glu Thr Ile Asn Glu Glu Ala Ala Glu Trp 65 70 75 80 Asp Arg Leu His Pro Val His Ala Gly Pro Ile Ala Pro Gly Gln Met 85 90 95 Arg Asp Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu Gln 100 105 110 Glu Gln Ile Gly Trp Met Thr His Asn Pro Pro Ile Pro Val Gly Glu 115 120 125 Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val Arg Met 130 135 140 Tyr Ser Pro Thr Ser Ile Leu Asp Ile Arg Gln Gly Pro Lys Glu Pro 145 150 155 160 Phe Arg Asp Tyr Val Asp Arg Phe Tyr Lys Thr Leu Arg Ala Glu Gln 165 170 175 Ala Ser Gln Glu Val Lys Asn Trp Met Thr Glu Thr Leu Leu Val Gln 180 185 190 Asn Ala Asn Pro Asp Cys Lys Thr Ile Leu Lys Ala Leu Gly Pro Gly 195 200 205 Ala Thr Leu Glu Glu Met Met Thr Ala Cys Gln Gly Val Gly Gly Pro 210 215 220 Gly His Lys Ala Arg Val Leu 225 230 <210> 8 <211> 111 <212> PRT <213> Human immunodeficiency virus type 1 gp41 <400> 8 Gly Ala Ala Ser Met Thr Leu Thr Val Gln Ala Arg Gln Leu Leu Ser 1 5 10 15 Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala Ile Glu Ala Gln 20 25 30 Gln His Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu Gln Ala 35 40 45 Arg Ile Leu Ala Val Glu Arg Tyr Leu Lys Asp Gln Gln Leu Leu Gly 50 55 60 Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Ala Val Pro Trp 65 70 75 80 Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn Asn Met 85 90 95 Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu 100 105 110 <210> 9 <211> 21 <212> PRT <213> Human immunodeficiency virus type 2 gp36 <400> 9 Asp Gln Ala Arg Leu Asn Ser Trp Gly Cys Ala Phe Arg Gln Val Cys 1 5 10 15 His Thr Thr Val Asn 20 <210> 10 <211> 96 <212> PRT <213> Human immunodeficiency virus type 1 group 0 gp41 <400> 10 Ile Gln Ala Gln Gln Gln Leu Leu Arg Leu Ser Val Trp Gly Ile Arg 1 5 10 15 Gln Leu Arg Ala Arg Leu Leu Ala Val Glu Thr Phe Leu Gln Asn Gln 20 25 30 Gln Leu Leu Ser Leu Trp Gly Cys Lys Gly Lys Leu Ile Cys Tyr Thr 35 40 45 Ser Val Gln Trp Asn Ala Ser Trp Gly Gly Asn Glu Ser Ile Trp Asp 50 55 60 Asn Leu Thr Trp Gln Glu Trp Asp Gln Gln Val Ser His Ile Ser Ser 65 70 75 80 Thr Ile Phe Glu Glu Ile Gln Lys Ala Gln Val Gln Gln Glu Gln Asn 85 90 95 <210> 11 <211> 484 <212> PRT <213> Artificial Sequence <220> <223> Combo amino acid sequence <400> 11 Met Pro Ile Val Gln Asn Leu Gln Gly Gln Met Val His Gln Ala Ile 1 5 10 15 Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val Val Glu Glu Lys Ala 20 25 30 Phe Ser Pro Glu Val Ile Pro Met Phe Ser Ala Leu Ser Glu Gly Ala 35 40 45 Thr Pro Gln Asp Leu Asn Thr Met Leu Asn Thr Val Gly Gly His Gln 50 55 60 Ala Ala Met Gln Met Leu Lys Glu Thr Ile Asn Glu Glu Ala Ala Glu 65 70 75 80 Trp Asp Arg Leu His Pro Val His Ala Gly Pro Ile Ala Pro Gly Gln 85 90 95 Met Arg Asp Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu 100 105 110 Gln Glu Gln Ile Gly Trp Met Thr His Asn Pro Pro Ile Pro Val Gly 115 120 125 Glu Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val Arg 130 135 140 Met Tyr Ser Pro Thr Ser Ile Leu Asp Ile Arg Gln Gly Pro Lys Glu 145 150 155 160 Pro Phe Arg Asp Tyr Val Asp Arg Phe Tyr Lys Thr Leu Arg Ala Glu 165 170 175 Gln Ala Ser Gln Glu Val Lys Asn Trp Met Thr Glu Thr Leu Leu Val 180 185 190 Gln Asn Ala Asn Pro Asp Cys Lys Thr Ile Leu Lys Ala Leu Gly Pro 195 200 205 Gly Ala Thr Leu Glu Glu Met Met Thr Ala Cys Gln Gly Val Gly Gly 210 215 220 Pro Gly His Lys Ala Arg Val Leu Gly Ser Gly Gly Gly Gly Gly Gly 225 230 235 240 Gly Ala Ala Ser Met Thr Leu Thr Val Gln Ala Arg Gln Leu Leu Ser 245 250 255 Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala Ile Glu Ala Gln 260 265 270 Gln His Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu Gln Ala 275 280 285 Arg Ile Leu Ala Val Glu Arg Tyr Leu Lys Asp Gln Gln Leu Leu Gly 290 295 300 Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Ala Val Pro Trp 305 310 315 320 Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn Asn Met 325 330 335 Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu Asp 340 345 350 Gln Ala Arg Leu Asn Ser Trp Gly Cys Ala Phe Arg Gln Val Cys His 355 360 365 Thr Thr Val Asn Glu Phe Gly Gly Gly Gly Gly Gly Ile Gln Ala Gln 370 375 380 Gln Gln Leu Leu Arg Leu Ser Val Trp Gly Ile Arg Gln Leu Arg Ala 385 390 395 400 Arg Leu Leu Ala Val Glu Thr Phe Leu Gln Asn Gln Gln Leu Leu Ser 405 410 415 Leu Trp Gly Cys Lys Gly Lys Leu Ile Cys Tyr Thr Ser Val Gln Trp 420 425 430 Asn Ala Ser Trp Gly Gly Asn Glu Ser Ile Trp Asp Asn Leu Thr Trp 435 440 445 Gln Glu Trp Asp Gln Gln Val Ser His Ile Ser Ser Thr Ile Phe Glu 450 455 460 Glu Ile Gln Lys Ala Gln Val Gln Gln Glu Gln Asn Leu Glu His His 465 470 475 480 His His His His<110> SUNG Jae Gap; LG CHEM INVESTMENT, LTD. <120> Recombinant combo protein fused in HIV type-1, -2 and 0, manufacturing method according, vector and transformant particular, diagnostic kit etc and composition for vaccines <130> NK-0251 <160> 11 <170> Kopatent In 1.71 <210> 1 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer 1 <400> 1 aaacatatgc agaacctcca ggggcaa 27 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer 2 <400> 2 aaaggatccc aaaactcttg ctttatg 27 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer 3 <400> 3 aaaggatccg gcgcagcgtc aatgacg 27 < 210> 4 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> Primer 4 <400> 4 aaagaattcg ttaacagtag tgtggcaaac ctgacggaaa gcgcaacccc aggagttcag 60 acgagcctgg tctaagcttg tgtaattgtt 90 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer 5 <400> 5 aaagaattca tacaggccca gcagcaa 27 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer 6 <400> 6 aaactcgaga ttttgttcct gctgtac 27 <210> 7 <211> 231 <212> PRT <213> Human immunodeficiency virus type 1 p24 <400> 7 Pro Ile Val Gln Asn Leu Gln Gly Gln Met Val His Gln Ala Ile Ser 1 5 10 15 Pro Arg Thr Leu Asn Ala Trp Val Lys Val Val Glu Glu Lys Ala Phe 20 25 30 Ser Pro Glu Val Ile Pro Met Phe Ser Ala Leu Ser Glu Gly Ala Thr 35 40 45 Pro Gln Asp Leu Asn Thr Met Leu Asn Thr Val Gly Gly His Gln Ala 50 55 60 Ala Met Gln Met Leu Lys Glu Thr Ile Asn Glu Glu Ala Ala Glu Trp 65 70 75 80 Asp Arg Leu His Pro Val His Ala Gly Pro Ile Ala Pro Gly Gln Met 85 90 95 Arg Asp Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu Gln 100 105 110 Glu Gln Ile Gly Trp Met Thr His Asn Pro Pro Ile Pro Val Gly Glu 115 120 125 Ile Tyr Lys Arg Trp Ile Lele Glu Leu Asn Lys Ile Val Arg Met 130 135 140 Tyr Ser Pro Thr Ser Ile Leu Asp Ile Arg Gln Gly Pro Lys Glu Pro 145 150 155 160 Phe Arg Asp Tyr Val Asp Arg Phe Tyr Lys Thr Leu Arg Ala Glu Gln 165 170 175 Ala Ser Gln Glu Val Lys Asn Trp Met Thr Glu Thr Leu Leu Val Gln 180 185 190 Asn Ala Asn Pro Asp Cys Lys Thr Ile Leu Lys Ala Leu Gly Pro Gly 195 200 205 Ala Thr Leu Glu Glu Met Met Thr Ala Cys Gln Gly Val Gly Gly Pro 210 215 220 Gly His Lys Ala Arg Val Leu 225 230 <210> 8 <211> 111 <212> PRT <213> Human immunodeficiency virus type 1 gp41 <400> 8 Gly Ala Ala Ser Met Thr Leu Thr Val Gln Ala Arg Gln Leu Leu Ser 1 5 10 15 Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala Ile Glu Ala Gln 20 25 30 Gln His Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu Gln Ala 35 40 45 Arg Ile Leu Ala Val Glu Arg Tyr Leu Lys Asp Gln Gln Leu Leu Gly 50 55 60 Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Ala Val Pro Trp 65 70 75 80 Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn Asn Met 85 90 95 Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu 100 105 110 <210> 9 <211> 21 <212> PRT <213> Human immunodeficiency virus type 2 gp36 <400> 9 Asp Gln Ala Arg Leu Asn Ser Trp Gly Cys Ala Phe Arg Gln Val Cys 1 5 10 15 His Thr Thr Val Val Asn 20 <210> 10 <211> 96 <212> PRT <213> Human immunodeficiency virus type 1 group 0 gp41 <400> 10 Ile Gln Ala Gln Gln Gln Leu Leu Arg Leu Ser Val Trp Gly Ile Arg 1 5 10 15 Gln Leu Arg Ala Arg Leu Leu Ala Val Glu Thr Phe Leu Gln Asn Gln 20 25 30 Gln Leu Leu Ser Leu Trp Gly Cys Lys Gly Lys Leu Ile Cys Tyr Thr 35 40 45 Ser Val Gln Trp Asn Ala Ser Trp Gly Gly Asn Glu Ser Ile Trp Asp 50 55 60 Asn Leu Thr Trp Gln Glu Trp Asp Gln Gln Val Ser His Ile Ser Ser 65 70 75 80 Thr Ile Phe Glu Glu Ile Gln Lys Ala Gln Val Gln Gln Glu Gln Asn 85 90 95 <210> 11 <211> 484 < 212> PRT <213> Artificial Sequence <220> <223> Combo amino acid sequence <400> 11 Met Pro Ile Val Gln Asn Leu Gln Gly Gln Met Val His Gln Ala Ile 1 5 10 15 Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val Val Glu Glu Lys Ala 20 25 30 Phe Ser Pro Glu Val Ile Pro Met Phe Ser Ala Leu Ser Glu Gly Ala 35 40 45 Thr Pro Gln Asp Leu Asn Thr Met Leu Asn Thr Val Gly Gly His Gln 50 55 60 Ala Ala Met Gln Met Leu Lys Glu Thr Ile Asn Glu Glu Ala Ala Glu 65 70 75 80 Trp Asp Arg Leu His Pro Val His Ala Gly Pro Ile Ala Pro Gly Gln 85 90 95 Met Arg Asp Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Ser Le Le 100 100 110 110 Gln Glu Gln Ile Gly Trp Met Thr His Asn Pro Pro Ile Pro Val Gly 115 120 125 Glu Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val Arg 130 135 140 Met Tyr Ser Pro Thr Ser Ile Leu Asp Ile Arg Gln Gly Pro Lys Glu 145 150 155 160 Pro Phe Arg Asp Tyr Val Asp Arg Phe Tyr Lys Thr Leu Arg Ala Glu 165 170 175 Gln Ala Ser Gln Glu Val Lys Asn Trp Met Thr Glu Thr Leu Leu Val 180 185 190 Gln Asn Ala Asn Pro Asp Cys Lys Thr Ile Leu Lys Ala Leu Gly Pro 195 200 205 Gly Ala Thr Leu Glu Glu M et Met Thr Ala Cys Gln Gly Val Gly Gly 210 215 220 Pro Gly His Lys Ala Arg Val Leu Gly Ser Gly Gly Gly Gly Gly Gly 225 230 235 240 Gly Ala Ala Ser Met Thr Leu Thr Val Gln Ala Arg Gln Leu Leu Ser 245 250 255 Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala Ile Glu Ala Gln 260 265 270 Gln His Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu Gln Ala 275 280 285 Arg Ile Leu Ala Val Glu Arg Tyr Leu Lys Asp Gln Gln Leu Leu Gly 290 295 300 Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Ala Val Pro Trp 305 310 315 320 Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn Asn Met 325 330 335 Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr S er Leu Asp 340 345 350 Gln Ala Arg Leu Asn Ser Trp Gly Cys Ala Phe Arg Gln Val Cys His 355 360 365 Thr Thr Val Asn Glu Phe Gly Gly Gly Gly Gly Gly Ile Gln Ala Gln 370 375 380 Gln Gln Leu Leu Arg Leu Ser Val Trp Gly Ile Arg Gln Leu Arg Ala 385 390 395 400 Arg Leu Leu Ala Val Glu Thr Phe Leu Gln Asn Gln Gln Leu Leu Ser 405 410 415 Leu Trp Gly Cys Lys Gly Lys Leu Ile Cys Tyr Thr Ser Val Gln Trp 420 425 430 Asn Ala Ser Trp Gly Gly Asn Glu Ser Ile Trp Asp Asn Leu Thr Trp 435 440 445 Gln Glu Trp Asp Gln Gln Val Ser His Ile Ser Ser Thr Ile Phe Glu 450 455 460 Glu Ile Gln Lys Ala Gln Val Gln Gln Glu Glu Gln Asn Leu Glu His His 465 470 475 480 His His His His

Claims (9)

삭제delete 인간 면역 결핍 바이러스(human immunodeficiency virus, 이하 'HIV'라 칭한다) 타입(type)-1 gp41 단백질(SEQ ID NO :8), HIV 타입(type)-2 gp36 단백질(SEQ ID NO :9) 및 HIV 타입(type)-1 그룹 O gp41 단백질(SEQ ID NO :10)을 포함하는 단백질의 N-말단에 HIV 타입(type)-1 p24 단백질(SEQ ID NO :7)이 결합된 것을 특징으로 하는 단일가닥 폴리펩티드인 융합단백질.Human immunodeficiency virus (hereinafter referred to as 'HIV') type-1 gp41 protein (SEQ ID NO: 8), HIV type-2 gp36 protein (SEQ ID NO: 9) and HIV The HIV type-1 p24 protein (SEQ ID NO: 7) is bound to the N-terminus of the protein containing the type-1 group O gp41 protein (SEQ ID NO: 10). A fusion protein that is a strand polypeptide. 제 2항에 있어서,The method of claim 2, 여섯번 반복되는 히스티딘 잔기가 상기 융합단백질의 C-말단에 위치하는 것을 특징으로 하는 상기 융합단백질.The fusion protein, characterized in that six times histidine residue is located at the C-terminus of the fusion protein. 제 3항의 상기 융합단백질을 암호화하는, 도 3에 기재된 개열지도를 갖는 발현벡터 pLCSK-1(기탁번호: KCTC 10100BP).An expression vector pLCSK-1 having a cleavage map as described in FIG. 3 encoding the fusion protein of claim 3 (Accession No .: KCTC 10100BP). 제 4항에 있어서,The method of claim 4, wherein 상기 발현벡터를 포함하는 것을 특징으로 하는 형질전환된 대장균(기탁번호: KCTC 10100BP).E. coli (transformation number: KCTC 10100BP) characterized in that it comprises the expression vector. 배양단계, 균체수집단계, 세포파쇄단계, 분리단계 및 정제단계를 포함하여 이루어지는 융합단백질의 생산방법에 있어서,In the production method of the fusion protein comprising a culturing step, cell collection step, cell disruption step, separation step and purification step, 제 5항의 상기 형질전환된 대장균을 이용하여 융합단백질을 생산하는 것을 특징으로 하는 융합단백질의 생산방법.Method for producing a fusion protein, characterized in that to produce a fusion protein using the transformed E. coli of claim 5. 제 6항에 있어서,The method of claim 6, 상기 정제단계가 융합단백질의 C말단에 있는 6개의 히스티틴 잔기를 프로본드칼럼에 흡착시켜 분리하는 방법에 의하여 이루어짐을 특징으로 하는 상기 융합단백질의 생산방법.Wherein the purification step is a method for producing the fusion protein, characterized in that by the method of separating by adsorbing six histitin residues at the C-terminal of the fusion protein to the probond column. 제 6항 또는 7항중 어느 한 항의 방법으로 생산된 단백질(SEQ ID NO :10)을 항원으로 사용하는 것을 특징으로 하는 HIV용 진단 키트.A diagnostic kit for HIV, which uses a protein (SEQ ID NO: 10) produced by the method of any one of claims 6 or 7 as an antigen. 삭제delete
KR10-2002-0000896A 2002-01-08 2002-01-08 Recombinant combo protein fused in HIV type-1 and -2, manufacturing method thereof, vector, transformant and diagnostic kit thereof KR100466382B1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
KR10-2002-0000896A KR100466382B1 (en) 2002-01-08 2002-01-08 Recombinant combo protein fused in HIV type-1 and -2, manufacturing method thereof, vector, transformant and diagnostic kit thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
KR10-2002-0000896A KR100466382B1 (en) 2002-01-08 2002-01-08 Recombinant combo protein fused in HIV type-1 and -2, manufacturing method thereof, vector, transformant and diagnostic kit thereof

Publications (2)

Publication Number Publication Date
KR20030060274A KR20030060274A (en) 2003-07-16
KR100466382B1 true KR100466382B1 (en) 2005-01-14

Family

ID=32217258

Family Applications (1)

Application Number Title Priority Date Filing Date
KR10-2002-0000896A KR100466382B1 (en) 2002-01-08 2002-01-08 Recombinant combo protein fused in HIV type-1 and -2, manufacturing method thereof, vector, transformant and diagnostic kit thereof

Country Status (1)

Country Link
KR (1) KR100466382B1 (en)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR900701836A (en) * 1988-10-03 1990-12-04 라메쉬 엘. 라탄 Peptides useful for diagnosing, preventing or treating new HIV proteins and AIDS
KR940000577A (en) * 1992-06-17 1994-01-03 김영길 Chimeric proteins useful for AIDS vaccines and methods for their preparation
KR950006463A (en) * 1993-08-27 1995-03-21 유충식 Sulfur HCV, HIV-1, HIV-2 and HTLV-1 Antibody Diagnostics
US5922533A (en) * 1997-08-15 1999-07-13 Abbott Laboratories Rapid assay for simultaneous detection and differentiation of antibodies to HIV groups
KR20010024287A (en) * 1997-09-26 2001-03-26 장 스테판느 Fusion proteins comprising hiv-1 tat and/or nef proteins
US6210874B1 (en) * 1988-01-27 2001-04-03 Biochem Immunosystems, Inc. Synthetic peptides and mixtures thereof for detecting HIV antibodies

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6210874B1 (en) * 1988-01-27 2001-04-03 Biochem Immunosystems, Inc. Synthetic peptides and mixtures thereof for detecting HIV antibodies
KR900701836A (en) * 1988-10-03 1990-12-04 라메쉬 엘. 라탄 Peptides useful for diagnosing, preventing or treating new HIV proteins and AIDS
KR940000577A (en) * 1992-06-17 1994-01-03 김영길 Chimeric proteins useful for AIDS vaccines and methods for their preparation
KR950006463A (en) * 1993-08-27 1995-03-21 유충식 Sulfur HCV, HIV-1, HIV-2 and HTLV-1 Antibody Diagnostics
US5922533A (en) * 1997-08-15 1999-07-13 Abbott Laboratories Rapid assay for simultaneous detection and differentiation of antibodies to HIV groups
KR20010024287A (en) * 1997-09-26 2001-03-26 장 스테판느 Fusion proteins comprising hiv-1 tat and/or nef proteins

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Detection and differentiation of HIV-1 group O sera from HIV-1 group M and HIV-2 using recombinant antigens and peptides Journal of Virological Methods 72(1998)43-49 43,44쪽 *

Also Published As

Publication number Publication date
KR20030060274A (en) 2003-07-16

Similar Documents

Publication Publication Date Title
US4839288A (en) Retrovirus capable of causing AIDS, antigens obtained from this retrovirus and corresponding antibodies and their application for diagnostic purposes
WO1994029339A1 (en) Tandem synthetic hiv-1 peptides
US5597896A (en) Retrovirus human immunodeficiency virus type 2(HIV2), capable of causing AIDS, antigens obtained from this retrovirus and corresponding antibodies and their application for diagnostic purposes
CN113018427A (en) Multivalent fusion protein vaccine based on neutralizing epitope of new coronavirus
US5268265A (en) Immunological complex comprising an antigen of Simian Immunodeficiency Virus (SIV) and an antibody against human immunodeficiency virus type 2 (HIV 2), and method and kit for detecting antibodies to HIV-2 reactive with antigens of SIV
Jeyarajah et al. Matrix-assisted laser desorption ionization/mass spectrometry mapping of human immunodeficiency virus-gp120 epitopes recognized by a limited polyclonal antibody
EP1417222B1 (en) Isolated polypeptides based on the neutralizing epitope of the p17 protein of hiv useful as vaccines, and neutralizing anti-p17 antibodies which specifically recognize said neutralizing epitope
WO2008034297A1 (en) Method of detecting antibodies against a series of human immunodeficiency virus proteins
KR100466382B1 (en) Recombinant combo protein fused in HIV type-1 and -2, manufacturing method thereof, vector, transformant and diagnostic kit thereof
JP4166940B2 (en) Synthetic antigens for detection of antibodies immunoreactive with HIV virus
US11237166B2 (en) Method for detecting HIV-1-specific antibodies using clade C env polypeptides
US6149910A (en) Peptides for the detection of HIV-1 group O
CA2676762C (en) Peptides for the detection of hiv-1 group o
Sohn et al. Overexpression and purification of human immunodeficiency virus type 1 env derived epitopes in Escherichia coli
WO1996027012A1 (en) Specific hiv-1 group o antigens
US5624795A (en) Isolation and characterization of a novel chimpanzee lentivirus, designated simian immunodeficiency virus isolate cpz-ant
CN113045671A (en) Recombinant antigen for detecting schistosoma japonicum, preparation method and application thereof
Ryabinina et al. Evaluation of the diagnostic efficiency of a recombinant antigen modeling the immunogenic epitope V3 of the envelope protein gp120 for immunoenzyme detection of antibodies to HIV-1
WO1989006240A1 (en) Peptide corresponding to hiv-env 583-599, and analogs thereof and applications of the peptides

Legal Events

Date Code Title Description
A201 Request for examination
N231 Notification of change of applicant
E902 Notification of reason for refusal
E701 Decision to grant or registration of patent right
GRNT Written decision to grant
FPAY Annual fee payment

Payment date: 20130102

Year of fee payment: 9

FPAY Annual fee payment

Payment date: 20131211

Year of fee payment: 10

FPAY Annual fee payment

Payment date: 20141217

Year of fee payment: 11

FPAY Annual fee payment

Payment date: 20151204

Year of fee payment: 12

FPAY Annual fee payment

Payment date: 20161212

Year of fee payment: 13

FPAY Annual fee payment

Payment date: 20180102

Year of fee payment: 14

FPAY Annual fee payment

Payment date: 20190102

Year of fee payment: 15

FPAY Annual fee payment

Payment date: 20200102

Year of fee payment: 16