KR20130136016A - Screening kit for human papillomavirus antibody using fusion polypeptide hpv antigen - Google Patents

Screening kit for human papillomavirus antibody using fusion polypeptide hpv antigen Download PDF

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KR20130136016A
KR20130136016A KR1020120053336A KR20120053336A KR20130136016A KR 20130136016 A KR20130136016 A KR 20130136016A KR 1020120053336 A KR1020120053336 A KR 1020120053336A KR 20120053336 A KR20120053336 A KR 20120053336A KR 20130136016 A KR20130136016 A KR 20130136016A
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안웅식
배수미
김용완
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주식회사 진코스
안웅식
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Abstract

The present invention relates to a kit for screening HPV antibody using a fusion polypeptide HPV antigen. More particularly, the present invention relates to a fusion polypeptide comprising an amino acid sequence of a Histidine tag at the N-terminal, an amino acid sequence of a Simian virus 40 large T-antigen at the C-terminal or its active fragment, and an amino acid sequence of an HPV L1 antigen selected from the group consisting of HPV 6 L1 antigen, HPV11 L1 antigen, HPV16 L1 antigen, and HPV18 L1 antigen, or its active fragment; a method for detecting an HPV antibody in a sample by using the fusion polypeptide HPV antigen; and an HPV antibody detecting kit for a bead array, comprising the fusion polypeptide HPV antigen. The use of the present invention can isolate and purify a recombinant HPV antigen at high purity without influencing activity to specifically bind to the HPV antibody and attach the recombinant HPV antigen to beads for a bead array effectively and uniformly, so that HPV 6, 11, 16, and 18 antibodies can be complexly detected in the sample with high sensitivity and accuracy. Further, the present invention can be utilized to determine the inoculation time of HPV vaccine and determine the defense period after inoculation by measuring the antibody titer.

Description

융합 폴리펩타이드 ＨＰＶ 항원을 이용한 ＨＰＶ 항체 스크리닝 키트{Screening Kit for Human Papillomavirus antibody using fusion polypeptide HPV antigen}Screening Kit for Human Papillomavirus antibody using fusion polypeptide HPV antigen}

본 발명은 융합 폴리펩타이드 HPV 항원을 이용한 HPV 항체 스크리닝 키트로서, N 말단에 히스티딘 택(Histidine tag)의 아미노산 서열, C 말단에 시미안 바이러스 40 대형 T 항원(simian virus 40 large T-antigen)의 아미노산 서열 또는 이의 활성단편, 및 HPV 6 L1 항원, HPV11 L1 항원, HPV16 L1 항원 및 HPV18 L1 항원으로 구성된 군으로부터 선택된 HPV L1 항원의 아미노산 서열 또는 이의 활성단편을 포함하는 융합 폴리펩타이드가 결합된 비드 어레이용 비드; 표지자 표지된 2차 항체; 및 형광물질이 부착된 상기 표지자의 특이적 결합제를 포함하는 비드 어레이용 HPV 항체 검출용 키트에 관한 것이다.
The present invention relates to an HPV antibody screening kit using a fusion polypeptide HPV antigen, comprising: an amino acid sequence of a histidine tag at the N-terminus and an amino acid of a simian virus 40 large T-antigen at the C-terminus. For bead arrays to which a fusion polypeptide comprising a sequence or active fragment thereof and an amino acid sequence of an HPV L1 antigen selected from the group consisting of HPV 6 L1 antigen, HPV11 L1 antigen, HPV16 L1 antigen and HPV18 L1 antigen or an active fragment thereof is bound Beads; Marker labeled secondary antibodies; And it relates to a kit for detecting bead array HPV antibody comprising a specific binding agent of the marker to which the fluorescent material is attached.

전세계적으로 매 2분마다 한 명의 여성이 자궁경부암으로 사망한다. 세계적으로 매년 발생하고 있는 자궁경부암은 전세계 여성에게 2번째로 빈번히 발생하는 암이며, 해마다 약 50만 명에게 발병하며 특히 여성에게는 유방암과 폐암에 이어 세 번째로 주된 암 사망의 원인이다. 자궁경부암의 99% 이상에서 HPV(Human Papillomavirus) 감염이 발견되는데, 이러한 HPV는 100종이 넘으며 그 중 30 ∼ 40 종은 점막조직(mucosal tissue) 감염을 유발하며 저위험성 HPV(low-risk HPV)와 발암성 HPV로 나뉜다. 무엇보다 중요한 것은, 자궁경부암과 직접 관련이 있는 15종 이상의 발암성 HPV이다. 이러한 발암성 HPV 중 16번, 18번이 가장 중요한데 이는 전세계적으로 70% 이상의 자궁경부암에서 발견된다. 대부분의 HPV 감염은 보통 6개월에서 2년 내에 자연 치유되지만 발암성 HPV에 지속적으로 감염되면 자궁경부암으로 발전할 수 있다.
Every two minutes, one woman dies of cervical cancer worldwide. Cervical cancer, which occurs worldwide annually, is the second most common cancer among women worldwide, affecting about half a million people each year, and among women, the third leading cause of cancer deaths after breast and lung cancer. Human Papillomavirus (HPV) infections are found in more than 99% of cervical cancers, including more than 100 HPVs, 30-40 of which cause mucosal tissue infections, and low-risk HPVs. And carcinogenic HPV. Most importantly, there are more than 15 carcinogenic HPVs that are directly linked to cervical cancer. Of these carcinogenic HPVs, 16 and 18 are the most important and are found in more than 70% of cervical cancers worldwide. Most HPV infections usually heal naturally within six months to two years, but persistent infection with carcinogenic HPV can lead to cervical cancer.

HPV의 아형의 종류는 인종적, 지형적, 환경적 특징을 갖기 때문에 백신개발에 있어 그 나라 고유의 아형 실정을 바로 알아야 하며, 한국에서 많이 발생하는 아형을 이용한 한국 고유의 백신 개발이 필요하며 나아가 이러한 백신의 개발시 필수적으로 백신의 면역원성(immunity)을 측정할 수 있는 기술 시스템이 확립되어야 한다. 현재까지 개발된 Gardasil(Merck사)와 Cervarix(GSK사)와 같은 HPV 예방백신 접종 후 항체의 유지기간과 효능 나아가 백신의 투여시기를 조사하기 위해서는 혈청 내 이들 HPV 아형에 대해 생성된 항체의 역가를 측정하는 것이 무엇보다 중요하다. 그러나 이러한 백신에 대한 면역원성을 동시에 측정할 수 있는 기법이 아직 확립되지 못했다. 최근에 개발된 마이크로칩 기술을 이용한 HPV 항체 스크리닝 키트(바이오메드랩, 한국)는 슬라이드상에서 반응하는 2차원적인 방법으로 교잡반응 후에 3차에 걸쳐서 세척과정을 거쳐야 하는 번거로움이 있다. 또한 서스펜션 어레이를 이용한 방법의 HPV 항체 스크리닝 키트가 개발되어 있으나, 신호 강도가 매우 낮아 현실적으로 스크리닝을 하기에는 그 한계가 있었다.Since the types of HPV subtypes have racial, geographical and environmental characteristics, it is necessary to know the specific subtype status of the country in developing vaccines, and it is necessary to develop Korea's own vaccines using many subtypes that occur in Korea. In the development of the system, it is essential that a technical system be established to measure the immunogenicity of the vaccine. To investigate the retention and efficacy of antibodies after HPV vaccinations, such as Gardasil (Merck) and Cervarix (GSK), which have been developed to date, and the timing of vaccine administration, the titers of antibodies produced against these HPV subtypes in serum are measured. It is important to measure. However, no technique has yet been established to simultaneously measure immunogenicity against these vaccines. Recently developed HPV antibody screening kit using microchip technology (BioMedLab, Korea) is a two-dimensional method of reacting on a slide, and has to be washed three times after the hybridization reaction. In addition, the HPV antibody screening kit of the method using a suspension array has been developed, but the signal strength is very low, there was a limit to the screening in reality.

이에, 본 발명자들은 백신의 면역원성을 측정할 수 있는 HPV 항체 스크리닝 기술을 확립하기 위해 노력한 결과, HPV 항원을 N 말단에 히스티딘 택(Histidine tag)의 아미노산 서열, C 말단에 시미안 바이러스 40 대형 T 항원(simian virus 40 large T-antigen)의 아미노산 서열 또는 이의 활성단편과 융합함으로써, HPV 항체에 특이적으로 결합할 수 있는 활성에 영향을 미치지 않으면서도 재조합 HPV 항원을 고순도로 분리, 정제하고 비드 어레이용 비드에 효과적으로 균일하게 부착시킬 수 있어 높은 민감도 및 정확도로 신속하게 시료 내 HPV 항체를 스크리닝할 수 있다는 것을 발견하고 본 발명을 완성하였다.
Accordingly, the present inventors have endeavored to establish an HPV antibody screening technique capable of measuring the immunogenicity of the vaccine. As a result, the amino acid sequence of the histidine tag at the N-terminus and the Simian virus 40 large T at the C-terminus are obtained. By fusion with the amino acid sequence of the antigen (simian virus 40 large T-antigen) or its active fragments, the recombinant HPV antigen can be isolated, purified and beaded in high purity without affecting the activity that can specifically bind to the HPV antibody. The present invention has been completed and found that it can be effectively and uniformly attached to the beads used to screen HPV antibodies in a sample quickly with high sensitivity and accuracy.

본 발명의 목적은 N 말단에 히스티딘 택(Histidine tag)의 아미노산 서열, C 말단에 시미안 바이러스 40 대형 T 항원(simian virus 40 large T-antigen)의 아미노산 서열 또는 이의 활성단편, 및 HPV 6 L1 항원, HPV11 L1 항원, HPV16 L1 항원 및 HPV18 L1 항원으로 구성된 군으로부터 선택된 HPV L1 항원의 아미노산 서열 또는 이의 활성단편을 포함하는 융합 폴리펩타이드가 결합된 비드 어레이용 비드; 표지자 표지된 2차 항체; 및 형광물질이 부착된 상기 표지자의 특이적 결합제를 포함하는 비드 어레이용 HPV 항체 검출용 키트를 제공하는 것이다.
An object of the present invention is an amino acid sequence of a histidine tag at the N terminus, an amino acid sequence of the simian virus 40 large T antigen at the C terminus, or an active fragment thereof, and an HPV 6 L1 antigen. Beads for arrays of beads bound to a fusion polypeptide comprising an amino acid sequence of an HPV L1 antigen selected from the group consisting of HPV11 L1 antigen, HPV16 L1 antigen and HPV18 L1 antigen, or an active fragment thereof; Marker labeled secondary antibodies; And it provides a kit for detecting bead array HPV antibody comprising a specific binding agent of the marker to which the fluorescent material is attached.

하나의 양태로서 본 발명은 N 말단에 히스티딘 택(Histidine tag)의 아미노산 서열; C 말단에 시미안 바이러스 40 대형 T 항원(simian virus 40 large T-antigen)의 아미노산 서열 또는 이의 활성단편; 및 HPV 6 L1 항원, HPV11 L1 항원, HPV16 L1 항원 및 HPV18 L1 항원으로 구성된 군으로부터 선택된 HPV L1 항원의 아미노산 서열 또는 이의 활성단편을 포함하는 융합 폴리펩타이드를 제공한다. 상기 융합 폴리펩타이드의 아미노산 서열은 서열번호 1 내지 4로 구성된 군으로부터 선택된 아미노산 서열일 수 있다. 상기 활성단편의 길이는 제한되지 않으나 본 발명의 구체적인 실시예에 따르면 시미안 바이러스 40 대형 T 항원 활성단편 길이는 11개 아미노산일 수 있다.
In one embodiment, the invention provides an amino acid sequence of a histidine tag at the N-terminus; The amino acid sequence of the simian virus 40 large T-antigen at its C terminus or an active fragment thereof; And an amino acid sequence of an HPV L1 antigen selected from the group consisting of HPV 6 L1 antigen, HPV11 L1 antigen, HPV16 L1 antigen and HPV18 L1 antigen, or an active fragment thereof. The amino acid sequence of the fusion polypeptide may be an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4. The length of the active fragment is not limited, but according to a specific embodiment of the present invention, the length of the Simian virus 40 large T antigen active fragment may be 11 amino acids.

HPV 항원을 N 말단에 히스티딘 택(Histidine tag)의 아미노산 서열, C 말단에 시미안 바이러스 40 대형 T 항원(simian virus 40 large T-antigen)의 아미노산 서열 또는 이의 활성단편과 융합함으로써, HPV 항체에 특이적으로 결합할 수 있는 활성에 영향을 미치지 않으면서도, 재조합 HPV 항원을 고순도로 분리, 정제하고 비드 어레이용 비드에 효과적으로 균일하게 부착시킬 수 있으며, 시미안 바이러스 40 대형 T 항원을 통하여 재조합 HPV 항원이 비드에 잘 부착되었는지, 부착시킨 양이 항상 동일한지, HPV 항원 4종간에 비드에 부착된 정도가 균일한지 등을 판정할 수 있어, 높은 민감도 및 정확도로 시료 내 HPV 항체를 스크리닝할 수 있다.
Specific for HPV antibodies by fusing the HPV antigen with the amino acid sequence of histidine tag at the N terminus, the amino acid sequence of simian virus 40 large T-antigen at the C terminus, or an active fragment thereof It is possible to isolate and purify recombinant HPV antigen with high purity and attach it effectively and uniformly to beads for bead arrays without affecting the ability to bind to them. It is possible to determine whether they adhere well to the beads, whether the amount of attachment is always the same, and whether the degree of adhesion to the beads between the four HPV antigens is uniform, and the like, to screen HPV antibodies in a sample with high sensitivity and accuracy.

또한, 상기 아미노산 서열과 하나 이상의 아미노산 잔기가 상이한 서열을 가지는 폴리펩타이드를 포함할 수 있다. 분자의 활성을 전체적으로 변경시키지 않는 단백질 및 폴리펩타이드에서의 아미노산 교환은 당해 분야에 공지되어 있다. 가장 통상적으로 일어나는 교환은 아미노산 잔기 Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly 간의 교환이다. 또한, 아미노산 서열상의 변이 또는 수식에 의해서 단백질의 열, pH 등에 대한 구조적 안정성이 증가하거나 단백질 활성이 증가한 단백질을 포함할 수 있다.
In addition, the amino acid sequence and one or more amino acid residues may include a polypeptide having a different sequence. Amino acid exchanges in proteins and polypeptides that do not alter the activity of the molecule as a whole are known in the art. The most commonly occurring exchanges are amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Exchange between Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly. In addition, the protein may include a protein having increased structural stability or increased protein activity against heat, pH, etc. of the protein by variation or modification on the amino acid sequence.

본 발명의 융합 폴리펩타이드는 당해 분야에 공지된 화학적 펩타이드 합성방법으로 제조하거나, 각 부위를 암호화하는 유전자를 PCR(polymerase chain reaction)에 의해 증폭하거나 공지된 방법으로 합성한 후 인프레임(in frame)으로 연결하여 발현벡터에 작동 가능하게 연결하여 클로닝하여 발현시킬 수 있다.
The fusion polypeptides of the present invention may be prepared by chemical peptide synthesis methods known in the art, or may be synthesized in a frame after amplifying genes encoding respective sites by PCR (polymerase chain reaction) or by known methods. It can be expressed by cloning by operably linked to the expression vector by linking.

또 하나의 양태로서 본 발명은 상기 융합 폴리펩타이드를 암호화하는 폴리뉴클레오티드, 상기 폴리뉴클레오티드를 포함하는 발현벡터, 상기 발현벡터로 형질전환된 형질전환체를 제공한다.In another aspect, the present invention provides a polynucleotide encoding the fusion polypeptide, an expression vector comprising the polynucleotide, and a transformant transformed with the expression vector.

상기 융합 폴리펩타이드를 암호화하는 폴리뉴클레오티드는 서열번호 5 내지 8로 구성된 군으로부터 선택된 뉴클레오티드 서열일 수 있다.
The polynucleotide encoding the fusion polypeptide may be a nucleotide sequence selected from the group consisting of SEQ ID NOs: 5-8.

본 발명의 구체예에서, 상기 HPV 6 L1 항원의 아미노산 서열 또는 이의 활성단편을 암호화하는 폴리뉴클레오티드를 pET-28a-SV40 벡터에 삽입하여 도 1에 개시된 개열지도를 가지는 pET-28a-SV40/HPV6 L1 벡터를 제조하는 단계, 상기 pET-28a-SV40/HPV6 L1 벡터를 Escherichia coli BL21 Gen-XTM에 형질전환하는 단계 및 상기 형질전환체로부터 HPV 6 L1 항원을 포함하는 융합 폴리펩타이드를 수득하는 단계를 통해 HPV 6 L1 항원을 포함하는 융합 폴리펩타이드를 제조할 수 있다.In an embodiment of the present invention, a polynucleotide encoding the amino acid sequence of the HPV 6 L1 antigen or an active fragment thereof is inserted into a pET-28a-SV40 vector to have a pET-28a-SV40 / HPV6 L1 having a cleavage map as shown in FIG. Preparing a vector, Escherichia the pET-28a-SV40 / HPV6 L1 vector A fusion polypeptide comprising HPV 6 L1 antigen may be prepared by transforming to coli BL21 Gen-XTM and obtaining a fusion polypeptide comprising HPV 6 L1 antigen from the transformant.

또한, 본 발명의 구체예에서, 상기 HPV 11 L1 항원의 아미노산 서열 또는 이의 활성단편을 암호화하는 폴리뉴클레오티드를 pET-28a-SV40 벡터에 삽입하여 도 2에 개시된 개열지도를 가지는 pET-28a-SV40/HPV11 L1 벡터를 제조하는 단계, 상기 pET-28a-SV40/HPV11 L1 벡터를 Escherichia coli BL21 Gen-XTM에 형질전환하는 단계 및 상기 형질전환체로부터 HPV 11 L1 항원을 포함하는 융합 폴리펩타이드를 수득하는 단계를 통해 HPV 11 L1 항원을 포함하는 융합 폴리펩타이드를 제조할 수 있다.In addition, in an embodiment of the present invention, a polynucleotide encoding the amino acid sequence of the HPV 11 L1 antigen or an active fragment thereof is inserted into a pET-28a-SV40 vector to have a pET-28a-SV40 / having a cleavage map as shown in FIG. 2. Preparing an HPV11 L1 vector, transforming the pET-28a-SV40 / HPV11 L1 vector into Escherichia coli BL21 Gen-XTM, and obtaining a fusion polypeptide comprising an HPV 11 L1 antigen from the transformant A fusion polypeptide comprising the HPV 11 L1 antigen can be prepared through.

또한, 본 발명의 구체예에서, 상기 HPV 16 L1 항원의 아미노산 서열 또는 이의 활성단편을 암호화하는 폴리뉴클레오티드를 pET-28a-SV40 벡터에 삽입하여 도 3에 개시된 개열지도를 가지는 pET-28a-SV40/HPV16 L1 벡터를 제조하는 단계, 상기 pET-28a-SV40/HPV16 L1 벡터를 Escherichia coli BL21 Gen-XTM에 형질전환하는 단계 및 상기 형질전환체로부터 HPV 16 L1 항원을 포함하는 융합 폴리펩타이드를 수득하는 단계를 통해 HPV 16 L1 항원을 포함하는 융합 폴리펩타이드를 제조할 수 있다.In addition, in an embodiment of the present invention, a polynucleotide encoding the amino acid sequence of the HPV 16 L1 antigen or an active fragment thereof is inserted into the pET-28a-SV40 vector to have a pET-28a-SV40 / having a cleavage map as shown in FIG. preparing an HPV16 L1 vector, wherein the pET-28a-SV40 / HPV16 L1 vector for Escherichia A fusion polypeptide comprising HPV 16 L1 antigen may be prepared by transforming to coli BL21 Gen-XTM and obtaining a fusion polypeptide comprising HPV 16 L1 antigen from the transformant.

또한, 본 발명의 구체예에서, 상기 HPV 18 L1 항원의 아미노산 서열 또는 이의 활성단편을 암호화하는 폴리뉴클레오티드를 pET-28a-SV40 벡터에 삽입하여 도 4에 개시된 개열지도를 가지는 pET-28a-SV40/HPV18 L1 벡터를 제조하는 단계, 상기 pET-28a-SV40/HPV18 L1 벡터를 Escherichia coli BL21 Gen-XTM에 형질전환하는 단계 및 상기 형질전환체로부터 HPV 18 L1 항원을 포함하는 융합 폴리펩타이드를 수득하는 단계를 통해 HPV 18 L1 항원을 포함하는 융합 폴리펩타이드를 제조할 수 있다.
In addition, in an embodiment of the present invention, a polynucleotide encoding the amino acid sequence of the HPV 18 L1 antigen or an active fragment thereof is inserted into the pET-28a-SV40 vector to have a pET-28a-SV40 / having a cleavage map as shown in FIG. 4. preparing an HPV18 L1 vector, wherein the pET-28a-SV40 / HPV18 L1 vector for Escherichia A fusion polypeptide comprising the HPV 18 L1 antigen may be prepared by transforming to coli BL21 Gen-X ™ and obtaining a fusion polypeptide comprising the HPV 18 L1 antigen from the transformant.

HPV 항원의 N 말단에 히스티딘 택(Histidine tag)의 아미노산 서열, C 말단에 시미안 바이러스 40 대형 T 항원(simian virus 40 large T-antigen)의 아미노산 서열 또는 이의 활성단편과 융합함으로써, 재조합 HPV 항원을 고순도로 분리, 정제하고 비드 어레이용 비드에 효과적으로 균일하게 부착시킬 수 있으며, 시미안 바이러스 40 대형 T 항원을 통하여 재조합 HPV 항원이 비드에 잘 부착되었는지, 부착시킨 양이 항상 동일한지, HPV 항원 4종간에 비드에 부착된 정도가 균일한지 등을 판정할 수 있는데, HPV 항체에 특이적으로 결합할 수 있는 항원성에 영향을 미치지 않기 위해서는 HPV 항원 부위의 선택, 결합 방향, 결합 위치가 모두 고려되어야 한다. 양 말단의 융합 부분의 존재로 인하여 항원성을 나타내는 부위가 분자 내부로 몰입되어 항원성이 감소하는 경우도 발생할 수 있기 때문이다. 본 발명의 융합 폴리펩타이드가 시료 내 HPV 항체를 높은 민감도 및 정확도로 스크리닝할 수 있다는 것은 전혀 유추할 수 없는 효과이다.
The recombinant HPV antigen is fused by fusion with the amino acid sequence of the histidine tag at the N terminus of the HPV antigen, the amino acid sequence of the simian virus 40 large T-antigen at the C terminus, or an active fragment thereof. Highly isolated and purified with high purity and can be effectively and uniformly attached to beads for bead arrays. It is possible to determine whether the degree of adhesion to the beads is uniform. In order not to affect the antigenicity that can specifically bind to HPV antibodies, the selection of the HPV antigen site, the binding direction, and the binding position should all be considered. This is because the presence of the fusion moiety at both ends may cause the site of antigenicity to be immersed in the molecule, thereby reducing the antigenicity. The fusion polypeptide of the present invention is capable of screening HPV antibodies in a sample with high sensitivity and accuracy is an inferior effect.

상기 폴리뉴클레오티드 서열은 하나 이상의 염기가 치환, 결실, 삽입 또는 이들의 조합에 의해 변이될 수 있다. 뉴클레오티드 서열을 화학적으로 합성하여 제조하는 경우, 당업계에 널리 공지된 합성법, 예를 들어 문헌(Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988)에 기술된 방법을 이용할 수 있으며, 트리에스테르, 포스파이트, 포스포르아미다이트 및 H-포스페이트 방법, PCR 및 기타 오토프라이머 방법, 고체 지지체상의올리고뉴클레오티드 합성법 등을 들 수 있다.
The polynucleotide sequence may be mutated by one or more bases substituted, deleted, inserted, or a combination thereof. When chemically synthesizing nucleotide sequences, synthetic methods well known in the art may be used, for example, those described in Engels and Uhlmann, Angew Chem Int Ed Eng., 37: 73-127, 1988. , Triester, phosphite, phosphoramidite and H-phosphate methods, PCR and other autoprimer methods, oligonucleotide synthesis on a solid support, and the like.

본 발명에서 "작동가능하게 연결(operably linked)"은 일반적 기능을 수행하도록 핵산 발현조절 서열과 목적하는 단백질 또는 RNA를 암호화하는 핵산 서열이 기능적으로 연결(functional linkage)되어 있는 것을 말한다. 예를 들어 프로모터와 단백질 또는 RNA를 암호화하는 핵산 서열이 작동가능하게 연결되어 코딩서열의 발현에 영향을 미칠 수 있다. 발현 벡터와의 작동적 연결은 당해 기술분야에서 잘 알려진 유전자 재조합 기술을 이용하여 제조할 수 있으며, 부위-특이적 DNA 절단 및 연결은 당해 기술 분야에서 일반적으로 알려진 효소 등을 사용한다. In the present invention, "operably linked" refers to a functional linkage of a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein or RNA of interest to perform a general function. For example, promoters and nucleic acid sequences encoding proteins or RNAs can be operably linked to affect expression of coding sequences. Operative linkage with expression vectors can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation employs enzymes and the like generally known in the art.

본 발명에서 "발현벡터"란 적당한 숙주세포에서 목적 단백질을 발현할 수 있는 재조합 벡터로서, 유전자 삽입물이 발현되도록 작동하게 연결된 필수적인 조절 요소를 포함하는 유전자 제작물을 말한다. 본 발명의 발현벡터는 적합한 발현벡터가 일반적으로 가지고 있는 요소로서 프로모터, 오퍼레이터, 개시코돈 같은 발현 조절 요소들을 포함한다. 개시 코돈 및 종결 코돈은 일반적으로 폴리펩타이드를 암호화하는 뉴클레오티드 서열의 일부로 간주되며, 유전자 제작물이 투여되었을 때 개체에서 반드시 작용을 나타내야 하며 코딩 서열과 인프레임(in frame)에 있어야 한다. 벡터의 프로모터는 구성적 또는 유도성일 수 있다. As used herein, an "expression vector" refers to a gene construct that is a recombinant vector capable of expressing a protein of interest in a suitable host cell and includes essential regulatory elements operably linked to express a gene insert. The expression vectors of the present invention include expression control elements such as promoters, operators, initiation codons as elements generally possessed by suitable expression vectors. The initiation codon and termination codon are generally considered to be part of the nucleotide sequence encoding the polypeptide and must be operative in the individual when the gene product is administered and in the coding sequence and in frame. The promoter of the vector may be constitutive or inducible.

또한, 세포 배양액으로부터 단백질의 분리를 촉진하기 위하여 융합 폴리펩타이드의 배출을 위한 시그널 서열을 포함할 수 있다. 특이적인 개시 시그널은 또한 삽입된 핵산 서열의 효율적인 번역에 필요할 수도 있다. 이들 시그널은 ATG 개시 코돈 및 인접한 서열들을 포함한다. 어떤 경우에는, ATG 개시 코돈을 포함할 수 있는 외인성 번역 조절 시그널이 제공되어야 한다. 이들 외인성 번역 조절 시그널들 및 개시 코돈들은 다양한 천연 및 합성 공급원일 수 있다. 발현 효율은 적당한 전사 또는 번역 강화 인자의 도입에 의하여 증가될 수 있다.
It may also include a signal sequence for the release of the fusion polypeptide to facilitate the separation of the protein from the cell culture. Specific initiation signals may also be required for efficient translation of inserted nucleic acid sequences. These signals include ATG start codons and contiguous sequences. In some cases, an exogenous translational control signal must be provided that can include an ATG start codon. These exogenous translational control signals and initiation codons can be various natural and synthetic sources. Expression efficiency can be increased by the introduction of appropriate transcriptional or translation enhancing factors.

발현벡터는 통상의 모든 발현 벡터를 다 사용할 수 있다. 숙주 세포에 따라서 단백질의 발현량과 수식 등이 다르게 나타나므로, 목적에 가장 적합한 숙주세포를 선택하여 사용하면 된다. 본 발명의 구체예에서, 본 발명의 융합 폴리펩타이드를 암호화하는 폴리뉴클레오티드를 포함한 발현 벡터로서 pET-28a-SV40/HPV6 L1 벡터, pET-28a-SV40/HPV11 L1 벡터, pET-28a-SV40/HPV16 L1 벡터, pET-28a-SV40/HPV18 L1 벡터를 제조하였다. As the expression vector, all conventional expression vectors can be used. Depending on the host cell, the expression level and expression of the protein are different. Therefore, the host cell may be selected and used according to the purpose. In an embodiment of the invention, the pET-28a-SV40 / HPV6 L1 vector, the pET-28a-SV40 / HPV11 L1 vector, pET-28a-SV40 / HPV16 as an expression vector comprising a polynucleotide encoding the fusion polypeptide of the invention L1 vector, pET-28a-SV40 / HPV18 L1 vector was prepared.

상기 벡터가 발현되는 형질전환체를 영양배지에서 배양하면 유용한 단백질을 대량으로 제조, 분리 가능하다. 배지와 배양조건은 숙주 세포에 따라 관용되는 것을 적당히 선택하여 이용할 수 있다. 배양시 세포의 생육과 단백질의 대량 생산에 적합하도록 온도, 배지의 pH 및 배양시간 등의 조건들을 적절하게 조절하여야 한다. 본 발명에 따른 발현 벡터로 형질전환될 수 있는 숙주 세포는 원핵 세포를 포함하며, DNA의 도입효율이 높고, 도입된 DNA의 발현효율이 높은 숙주가 통상 사용된다. 세균, 예를 들어 에쉐리키아, 슈도모나스, 바실러스, 스트렙토마이세스와 같은 주지의 원핵 숙주들이 사용될 수 있는 숙주 세포의 예이다. 바람직하게는 대장균이 사용될 수 있다. 펩타이드의 발현은 유도인자 IPTG를 사용하여 발현을 유도할 수 있고, 유도시간은 단백질의 양을 최대화되게 조절할 수 있다. 본 발명에서, 재조합적으로 생산된 펩타이드는 배지 또는 세포 분해물로부터 회수될 수 있다. 펩타이드 발현에 사용된 세포는 동결-해동 반복, 음파처리, 기계적 파괴 또는 세포 분해제와 같은 다양한 물질적 또는 화학적 수단에 의해 파괴될 수 있으며, 통상적인 생화학 분리 기술에 의해서 분리. 정제 가능하다(Sambrook et al., Molecular Cloning: A laborarory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989; Deuscher, M., Guide to Protein Purification Methods Enzymology, Vol. 182. Academic Press. Inc., San Diego, CA, 1990). 전기영동, 원심분리, 겔여과, 침전, 투석, 크로마토그래피(이온교화크로마토그래피, 친화력 크로마토그래피, 면역흡착 친화력 크로마토그래피, 역상 HPLC, 겔 침투 HPLC), 등전성 포커스 및 이의 다양한 변화 및 복합 방법을 포함하나 이에 국한되지 않는다. When the transformant expressing the vector is cultured in a nutrient medium, a large amount of useful protein can be prepared and separated. The medium and culture conditions may be appropriately selected depending on the host cell. In culture, conditions such as temperature, pH of the medium, and incubation time should be appropriately adjusted to be suitable for cell growth and mass production of proteins. Host cells that can be transformed with the expression vector according to the present invention include prokaryotic cells, and a host with high expression efficiency of introduced DNA and a high expression efficiency of introduced DNA is usually used. Bacteria, for example, known prokaryotic hosts such as Escherichia, Pseudomonas, Bacillus, Streptomyces, are examples of host cells that can be used. Preferably, Escherichia coli can be used. Expression of the peptide can be induced using inducer IPTG, and induction time can be controlled to maximize the amount of protein. In the present invention, recombinantly produced peptides can be recovered from medium or cell lysate. Cells used for peptide expression can be disrupted by a variety of physical or chemical means, such as freeze-thaw repeats, sonication, mechanical disruption or cell digestion, and are isolated by conventional biochemical separation techniques. Purification is possible (Sambrook et al., Molecular Cloning: A laborarory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989; Deuscher, M., Guide to Protein Purification Methods Enzymology, Vol. 182. Academic Press. Inc., San Diego, CA, 1990). Electrophoresis, centrifugation, gel filtration, precipitation, dialysis, chromatography (ion exchange chromatography, affinity chromatography, immunosorbent affinity chromatography, reverse phase HPLC, gel permeation HPLC), isoelectric focus and various variations and combinations thereof Including but not limited to.

또다른 양태로서, 본 발명은 상기 융합 폴리펩타이드를 비드 어레이용 비드에 결합시키는 단계; 상기 융합 폴리펩타이드가 결합된 비드를 시료와 반응시키는 단계; 상기 시료를 표지자 표지된 2차 항체와 반응시키는 단계; 상기 2차 항체를 형광물질이 부착된 상기 표지자의 특이적 결합제와 반응시키는 단계 및 비드 유래의 형광값과 표지자의 특이적 결합제 유래의 형광값을 측정하여 시료 내 HPV 항체를 검출하는 방법을 제공한다. 상기 표지자는 비오틴일 수 있고, 상기 표지자의 특이적 결합제는 스트렙타비딘일 수 있으며, 상기 형광물질은 플루오레신, 이소티오시아네이트, 로다민, 피코에리트린, 피코시아닌, 알로피코시아닌, o-프탈데히드 또는 플루오레스카민일 수 있다. 상기 비드 어레이는 다중 비드 어레이일 수 있고, 본 발명의 구체적인 실시예에서는 루미넥스 분석법을 이용하였다.
In another aspect, the present invention provides a method for preparing a bead array comprising: binding the fusion polypeptide to beads for beads array; Reacting the beads to which the fusion polypeptide is bound with a sample; Reacting the sample with a marker labeled secondary antibody; A method of detecting HPV antibodies in a sample by reacting the secondary antibody with a specific binding agent of the marker to which a fluorescent substance is attached and measuring a fluorescence value derived from a bead and a fluorescence value derived from a specific binding agent of a marker are provided. . The marker may be biotin, the specific binding agent of the marker may be streptavidin, the fluorescent material may be fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde or fluorescarmine. The bead array may be a multi-bead array, in the specific embodiment of the present invention was used luminex analysis.

본 발명에 따른 방법은 N 말단에 히스티딘 택(Histidine tag)의 아미노산 서열; C 말단에 시미안 바이러스 40 대형 T 항원(simian virus 40 large T-antigen)의 아미노산 서열 또는 이의 활성단편; 및 HPV 6 L1 항원, HPV11 L1 항원, HPV16 L1 항원 및 HPV18 L1 항원으로 구성된 군으로부터 선택된 HPV L1 항원의 아미노산 서열 또는 이의 활성단편을 포함하는 융합 폴리펩타이드인 재조합 HPV 항원을 비드 어레이용 비드에 결합시키는 단계를 포함한다.
The method according to the invention comprises an amino acid sequence of a histidine tag at the N terminus; The amino acid sequence of the simian virus 40 large T-antigen at its C terminus or an active fragment thereof; And a recombinant HPV antigen, which is a fusion polypeptide comprising an amino acid sequence of an HPV L1 antigen selected from the group consisting of HPV 6 L1 antigen, HPV11 L1 antigen, HPV16 L1 antigen and HPV18 L1 antigen, or an active fragment thereof, to beads for beads array. Steps.

상기 비드 어레이용 비드로 루미넥스 비드(Luminex bead)를 사용할 수 있다. 상기 비드는 다중 비드 어레이 방법에 사용될 수 있는 것이면 특별히 제한되지 않는다. 다중 비드 어레이용 비드는 색으로 100종류까지 구별할 수 있으므로 특정 색(번호)의 비드에 특정 항원을 붙여주고 여러 비드 세트를 섞어서 반응을 진행한 후 다중 비드 어레이 분석기(예를 들어, 루미넥스 분석기)로 각 비드의 색과 그 비드 표면의 항원 항체 반응량을 동시에 측정하면, HPV 6 L1 항체, HPV11 L1 항체, HPV16 L1 항체 또는 HPV18 L1 항체를 포함하여 여러 항체를 동시에 측정할 수 있다. 따라서 소량의 시료만으로 분석이 가능할 뿐만 아니라 1회 실험으로 여러 번의 실험을 대신할 수 있으므로 시간과 노동력이 절약된다.
Luminex beads may be used as beads for the bead array. The beads are not particularly limited as long as they can be used in the multiple bead array method. Since beads for multiple bead arrays can be distinguished up to 100 types by color, multiple bead array analyzers (for example, luminex analyzers) can be attached after attaching specific antigens to specific color (number) beads and mixing several bead sets. By simultaneously measuring the color of each bead and the reaction amount of the antigenic antibody on the surface of the bead, several antibodies can be measured simultaneously, including HPV 6 L1 antibody, HPV11 L1 antibody, HPV16 L1 antibody or HPV18 L1 antibody. Therefore, not only can a small sample be analyzed, but a single experiment can replace several experiments, saving time and labor.

또한, 본 발명에 따른 방법은 상기 융합 폴리펩타이드가 결합된 비드를 시료와 반응시키는 단계를 포함한다. 상기 시료는 HPV에 의하여 감염될 수 있는 개체들[예, 포유 동물 예컨대, 인간, 가축(예, 소, 말, 돼지, 양 및 염소) 및 애완 동물(예, 고양이 및 개)]등을 포함하는 개체에서 유래한다. 시료는 혈청, 세포 등 HPV 항체를 포함할 수 있는 것이면 모두 포함된다. 융합 폴리펩타이드 항원은 시료 내 HPV 6 L1 항체, HPV11 L1 항체, HPV16 L1 항체 및 HPV18 L1 항체와 항원-항체 반응을 한다.
In addition, the method according to the invention comprises the step of reacting the beads with the fusion polypeptide is bound to the sample. The sample may include individuals that may be infected by HPV (eg mammals such as humans, livestock (eg cattle, horses, pigs, sheep and goats) and pets (eg cats and dogs)). It comes from an individual. Samples are included as long as they can contain HPV antibodies, such as serum and cells. The fusion polypeptide antigens undergo antigen-antibody reactions with HPV 6 L1 antibodies, HPV11 L1 antibodies, HPV16 L1 antibodies and HPV18 L1 antibodies in the sample.

또한 본 발명은 상기 시료를 표지자 표지된 2차 항체와 반응시키는 단계 상기 2차 항체를 형광물질이 부착된 상기 표지자의 특이적 결합제와 반응시키는 단계 및 비드 유래의 형광값과 표지자의 특이적 결합제 유래의 형광값을 측정하여 시료 내 HPV 항체를 검출하는 단계를 포함한다. 상기 표지자는 비오틴(biotin)일 수 있고, 표지자의 특이적 결합제는 스트렙타비딘일 수 있다.
In another aspect, the present invention is the step of reacting the sample with a marker-labeled secondary antibody reacting the secondary antibody with a specific binding agent of the marker to which the fluorescent material is attached and the fluorescence value from the beads and the specific binding agent of the marker Detecting the HPV antibody in the sample by measuring the fluorescence value of the sample. The marker may be biotin and the specific binding agent of the marker may be streptavidin.

상기 2차 항체는 시료 내 HPV 6 L1 항체, HPV11 L1 항체, HPV16 L1 항체 및 HPV18 L1 항체와 특이적으로 결합하며 2차 항체에 결합된 비오틴은 스트렙타비딘(또는 아비딘)과 특이적으로 결합하므로 스트렙타비딘에 결합된 형광물질에서 형광을 발현하게 된다. 상기 형광물질은 예를 들어 플루오레신, 이소티오시아네이트, 로다민, 피코에리트린, 피코시아닌, 알로피코시아닌, o-프탈데히드 또는 플루오레스카민일 수 있으나 이에 제한되지 않으며 당업계에 공지된 다양한 형광물질을 사용할 수 있다.
The secondary antibody specifically binds HPV 6 L1 antibody, HPV11 L1 antibody, HPV16 L1 antibody and HPV18 L1 antibody in the sample, and biotin bound to the secondary antibody specifically binds to streptavidin (or avidin). Fluorescence is expressed in the fluorescent material bound to streptavidin. The fluorescent material may be, for example, but not limited to, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde or fluorescamine. Various known fluorescent materials can be used.

비드 유래의 형광값(각 비드의 색)과 표지자의 특이적 결합제 유래의 형광값을 함께 측정하여 항원 항체 반응량을 결정함으로써 시료 내 HPV 6 L1 항체, HPV11 L1 항체, HPV16 L1 항체 또는 HPV18 L1 항체의 존부 및 항체의 역가를 정확히 검출할 수 있다. 항체의 역가는 형광값의 판정 기준치(cutoff value) 설정하여 측정한다.
By measuring the fluorescence value derived from the beads (color of each bead) and the fluorescence value derived from the specific binding agent of the marker, the antigen antibody reaction amount was determined to determine the HPV 6 L1 antibody, HPV11 L1 antibody, HPV16 L1 antibody, or HPV18 L1 antibody in the sample. The presence of and the titer of the antibody can be detected accurately. The titer of an antibody is measured by setting the cutoff value of a fluorescence value.

본 발명의 구체적 실시예에서, 상기 HPV 항원을 N 말단에 히스티딘 택(Histidine tag)의 아미노산 서열, C 말단에 시미안 바이러스 40 대형 T 항원(simian virus 40 large T-antigen)의 아미노산 서열 또는 이의 활성단편과 융합함으로써, HPV 항체에 특이적으로 결합할 수 있는 활성에 영향을 미치지 않으면서도, 재조합 HPV 항원을 고순도로 분리, 정제하고 비드 어레이용 비드에 효과적으로 균일하게 부착시킬 수 있으며, 시미안 바이러스 40 대형 T 항원을 통하여 재조합 HPV 항원이 비드에 잘 부착되었는지, 부착시킨 양이 항상 동일한지, HPV 항원 4종간에 비드에 부착된 정도가 균일한지 등을 판정할 수 있어, 높은 민감도 및 정확도로 시료 내 HPV 6 L1 항체, HPV11 L1 항체, HPV16 L1 항체 또는 HPV18 L1 항체를 포함하는 HPV 항체를 스크리닝할 수 있었다.
In a specific embodiment of the invention, the amino acid sequence of the histidine tag (Histidine tag) at the N-terminal, the amino acid sequence of simian virus 40 large T-antigen at the C-terminal or its activity By fusion with fragments, recombinant HPV antigens can be isolated and purified in high purity and effectively attached uniformly to beads for bead arrays without affecting the ability to specifically bind HPV antibodies. Through the large T antigen, it is possible to determine whether the recombinant HPV antigen is well attached to the beads, whether the amount of attachment is always the same, and whether the degree of adhesion to the beads is uniform among the four HPV antigens, and so on. HPV antibodies can be screened including HPV 6 L1 antibodies, HPV11 L1 antibodies, HPV16 L1 antibodies or HPV18 L1 antibodies.

다른 양태로 본 발명은 N 말단에 히스티딘 택(Histidine tag)의 아미노산 서열, C 말단에 시미안 바이러스 40 대형 T 항원(simian virus 40 large T-antigen)의 아미노산 서열 또는 이의 활성단편, 및 HPV 6 L1 항원, HPV11 L1 항원, HPV16 L1 항원 및 HPV18 L1 항원으로 구성된 군으로부터 선택된 HPV L1 항원의 아미노산 서열 또는 이의 활성단편을 포함하는 융합 폴리펩타이드가 결합된 비드 어레이용 비드; 표지자 표지된 2차 항체; 및 형광물질이 부착된 상기 표지자의 특이적 결합제를 포함하는 비드 어레이용 HPV 항체 검출용 키트를 제공한다.
In another aspect, the present invention provides an amino acid sequence of histidine tag at the N terminus, an amino acid sequence of simian virus 40 large T-antigen at its C terminus, or an active fragment thereof, and HPV 6 L1. Beads for bead arrays to which a fusion polypeptide comprising an amino acid sequence of an HPV L1 antigen selected from the group consisting of an antigen, an HPV11 L1 antigen, an HPV16 L1 antigen, and an HPV18 L1 antigen or an active fragment thereof is bound; Marker labeled secondary antibodies; And it provides a kit for detecting the HPV antibody for the bead array comprising a specific binding agent of the marker to which the fluorescent material is attached.

본 발명을 이용하면 HPV 항체에 특이적으로 결합할 수 있는 활성에 영향을 미치지 않으면서도 재조합 HPV 항원을 고순도로 분리, 정제하고 비드 어레이용 비드에 효과적으로 균일하게 부착시킬 수 있어 높은 민감도 및 정확도로 시료 내 HPV 6, 11, 16 및 18 항체를 복합적으로 검출할 수 있다. 항체 역가를 측정하여 HPV 백신의 접종시기를 결정하는데 활용될 수 있고, 백신 접종 후 방어 기간의 측정 등에 활용 가능하다.
Using the present invention, it is possible to isolate and purify recombinant HPV antigens with high purity and effectively and uniformly attach them to beads for beads array without affecting the activity that can specifically bind to HPV antibodies. Internal HPV 6, 11, 16 and 18 antibodies can be detected in combination. The antibody titer can be used to determine the inoculation time of the HPV vaccine, and can be used to measure the duration of defense after vaccination.

도 1은 pET-28a-SV40/HPV6 L1 벡터를 나타낸 개열지도이다.
도 2는 pET-28a-SV40/HPV11 L1 벡터를 나타낸 개열지도이다.
도 3은 pET-28a-SV40/HPV16 L1 벡터를 나타낸 개열지도이다.
도 4는 pET-28a-SV40/HPV18 L1 벡터를 나타낸 개열지도이다.
도 5는 재조합 균주 E. coli BL21 pET-28a-SV40/HPV6 L1으로부터 발현한 HPV 6 L1 항원 포함 융합 폴리펩타이드를 Goat anti-mouse IgG-HRP항체를 사용하여 웨스턴 블랏팅한 것을 나타낸 것이다.
도 6은 재조합 균주 E. coli BL21 pET-28a-SV40/HPV11 L1으로부터 발현한 HPV 11 L1 항원 포함 융합 폴리펩타이드를 Goat anti-mouse IgG-HRP항체를 사용하여 웨스턴 블랏팅한 것을 나타낸 것이다.
도 7은 재조합 균주 E. coli BL21 pET-28a-SV40/HPV16 L1으로부터 발현한 HPV 16 L1 항원 포함 융합 폴리펩타이드를 Goat anti-mouse IgG-HRP항체를 사용하여 웨스턴 블랏팅한 것을 나타낸 것이다.
도 8은 재조합 균주 E. coli BL21 pET-28a-SV40/HPV18 L1으로부터 발현한 HPV 18 L1 항원 포함 융합 폴리펩타이드를 Goat anti-mouse IgG-HRP항체를 사용하여 웨스턴 블랏팅한 것을 나타낸 것이다. 1 is a cleavage map showing a pET-28a-SV40 / HPV6 L1 vector.
2 is a cleavage map showing the pET-28a-SV40 / HPV11 L1 vector.
3 is a cleavage map showing the pET-28a-SV40 / HPV16 L1 vector.
4 is a cleavage map showing the pET-28a-SV40 / HPV18 L1 vector.
FIG. 5 shows Western blotting of HPV 6 L1 antigen-containing fusion polypeptides expressed from recombinant strain E. coli BL21 pET-28a-SV40 / HPV6 L1 using Goat anti-mouse IgG-HRP antibody.
FIG. 6 shows Western blotting of HPV 11 L1 antigen-containing fusion polypeptides expressed from recombinant strain E. coli BL21 pET-28a-SV40 / HPV11 L1 using Goat anti-mouse IgG-HRP antibody.
FIG. 7 shows Western blotting of HPV 16 L1 antigen-containing fusion polypeptides expressed from recombinant strain E. coli BL21 pET-28a-SV40 / HPV16 L1 using Goat anti-mouse IgG-HRP antibody.
FIG. 8 shows Western blotting of HPV 18 L1 antigen-containing fusion polypeptides expressed from recombinant strain E. coli BL21 pET-28a-SV40 / HPV18 L1 using Goat anti-mouse IgG-HRP antibody.

이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.
Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

실시예1Example 1 : : HPVHPV 6  6 L1L1 , , HPVHPV 11  11 L1L1 , , HPVHPV 16  16 L1L1 및  And HPVHPV 18  18 L1L1 유전자의 발현을 위한 발현벡터의 구축 Construction of Expression Vectors for Gene Expression

독일로부터 분양 받은 recombinant plasmid harboring full genome of HPV 6, HPV 11, HPV 16 및 HPV 18 로부터 얻은 L1 유전자를 발현벡터에 삽입한 재조합 플라스미드(pET-28a-SV40/HPV6 L1, pET-28a-SV40/HPV11 L1, pET-28a-SV40/HPV16 L1 및 pET-28a-SV40/HPV18 L1)를 하기와 같이 구축하였다.Recombinant plasmid containing L1 gene from recombinant plasmid harboring full genome of HPV 6, HPV 11, HPV 16 and HPV 18 sold from Germany (pET-28a-SV40 / HPV6 L1, pET-28a-SV40 / HPV11) L1, pET-28a-SV40 / HPV16 L1 and pET-28a-SV40 / HPV18 L1) were constructed as follows.

먼저 HPV 6 L1, HPV 11 L1, HPV 16 L1 및 HPV 18 L1 유전자를 얻기 위해 HPV 6 L1 유전자의 염기서열로부터 N-말단 아미노산 서열을 암호화하는 서열번호 9 프라이머 (HPV6L1F) (5' 말단 프라이머)와 C-말단 아미노산 서열을 암호화하는 DNA 염기에 상보적인 서열번호 10 프라이머 (HPV6L1R) (3' 말단 프라이머) HPV 11 L1 유전자의 염기서열로부터 N-말단 아미노산 서열을 암호화하는 서열번호 11 프라이머 (HPV11L1F) (5' 말단 프라이머)와 C-말단 아미노산 서열을 암호화하는 DNA 염기에 상보적인 서열번호 12 프라이머 (HPV11L1R) (3' 말단 프라이머) HPV 16 L1 유전자의 염기서열로부터 N-말단 아미노산 서열을 암호화하는 서열번호 13 프라이머 (HPV16L1F) (5' 말단 프라이머)와 C-말단 아미노산 서열을 암호화하는 DNA 염기에 상보적인 서열번호 14 프라이머 (HPV16L1R) (3' 말단 프라이머) HPV 18 L1 유전자의 염기서열로부터 N-말단 아미노산 서열을 암호화하는 서열번호 15 프라이머 (HPV18L1F) (5' 말단 프라이머)와 C-말단 아미노산 서열을 암호화하는 DNA 염기에 상보적인 서열번호 16 프라이머 (HPV18L1R) (3' 말단 프라이머)를 각각 제작하였다. First, the SEQ ID NO: 9 primer (HPV6L1F) (5 'terminal primer) encoding the N-terminal amino acid sequence from the nucleotide sequence of the HPV 6 L1 gene to obtain HPV 6 L1, HPV 11 L1, HPV 16 L1 and HPV 18 L1 gene SEQ ID NO: 10 primer (HPV6L1R) (3 'terminal primer) complementary to the DNA base encoding the C-terminal amino acid sequence SEQ ID NO: 11 primer (HPV11L1F), which encodes the N-terminal amino acid sequence from the nucleotide sequence of the HPV 11 L1 gene ( SEQ ID NO: 12 primer (HPV11L1R) (3 'terminal primer) complementary to the DNA base encoding the C-terminal amino acid sequence) and the C-terminal amino acid sequence; SEQ ID No. encoding the N-terminal amino acid sequence from the nucleotide sequence of the HPV 16 L1 gene. 13 primer (HPV16L1F) (5 'terminal primer) and SEQ ID NO: 14 primer (HPV16L1R) (3' terminal primer) complementary to the DNA base encoding the C-terminal amino acid sequence of the HPV 18 L1 gene SEQ ID NO: 15 primer (HPV18L1F) (5 'terminal primer) encoding the N-terminal amino acid sequence from the nucleotide sequence and SEQ ID NO. 16 primer (HPV18L1R) (3' terminal primer) complementary to the DNA base encoding the C-terminal amino acid sequence ) Were produced respectively.

제작한 프라이머를 이용하여 각각의 HPV L1 유전자를 증폭시켰다. 서열번호 9, 11, 13, 15 프라이머는 제한효소 EcoRI 또는 SaiI 절단부위를, 서열번호 10, 12, 14, 16 프라이머는 제한효소 SalI 또는 HindIII 절단부위를 각각 포함하도록 합성하였다. Each HPV L1 gene was amplified using the prepared primers. The primers SEQ ID NO: 9, 11, 13, 15 were synthesized to contain the restriction enzyme EcoR I or Sai I cleavage site, and SEQ ID NO: 10, 12, 14, 16 primers to the restriction enzyme Sal I or Hind III cleavage site, respectively.

각각의 PCR은 0.2 ㎍ HPV 플라스미드 DNA와 10 pmole의 5'-말단 프라이머 및 3'-말단 프라이머, 200 μM dNTPs, 10X Accuprime DNA 중합효소 완충용액 및 2.5 U Accuprime DNA 중합효소 (invitrogen)로 이루어진 혼합 조성액을 95℃에서 3분간 반응시킨 후 95℃ 30초, 60℃에서 30초, 72℃에서 90초의 조건으로 35회 반복한 후, 마지막 72℃에서 5분간 반응시켰다.
Each PCR was a mixed composition consisting of 0.2 μg HPV plasmid DNA, 10 pmole 5'-terminal and 3'-terminal primers, 200 μM dNTPs, 10X Accuprime DNA polymerase buffer and 2.5 U Accuprime DNA polymerase (invitrogen). After reacting for 3 minutes at 95 ℃, repeated 35 times at the conditions of 95 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 90 seconds, and then reacted for 5 minutes at 72 ℃.

서열번호 9 프라이머 (HPV6L1F) :SEQ ID NO: 9 Primer (HPV6L1F):

5'- GAATTCATGTGGCGGCCTAGCGACAGC-3' (EcoRI 절단부위 포함)
5'- GAATTC ATGTGGCGGCCTAGCGACAGC-3 '(includes EcoR I cut)

서열번호 10 프라이머 (HPV6L1R) :SEQ ID NO: 10 Primer (HPV6L1R):

5'- GCATGAGTCGACCCTTTTAGTTTTGGCGCGC-3' (SalI 절단부위 포함)5'- GCATGA GTCGAC CCTTTTAGTTTTGGCGCGC-3 '(Including Sal I cut)

서열번호 11 프라이머 (HPV11L1F) :SEQ ID NO: 11 Primer (HPV11L1F):

5'- GAATTCATGTGGCGGCCTAGCGACAGC-3'(EcoRI 절단부위 포함)
5'- GAATTC ATGTGGCGGCCTAGCGACAGC-3 'with EcoR I cut

서열번호 12 프라이머 (HPV11L1R) :SEQ ID NO: 12 Primer (HPV11L1R):

5'- GCATGAGTCGACCTTTTTGGTTTTGGTACGTTT-3'(SalI 절단부위 포함)
5'- GCATGA GTCGAC CTTTTTGGTTTTGGTACGTTT-3 'with Sal I cut

서열번호 13 프라이머 (HPV16L1F) :SEQ ID NO: 13 Primer (HPV16L1F):

5'- GTCGACCGATGCAGGTGACTTTTATTTACATC-3'(SalI 절단부위 포함)
5'- GTCGAC CGATGCAGGTGACTTTTATTTACATC-3 'with Sal I cut

서열번호 14 프라이머 (HPV16L1R) :SEQ ID NO: 14 Primer (HPV16L1R):

5'- AAGCTTCAGCTTACGTTTTTTGCGTTTAGC-3' (HindⅢ절단부위 포함)
5'- AAGCTT CAGCTTACGTTTTTTGCGTTTAGC-3 '(including Hind III cutting site)

서열번호 15 프라이머 (HPV18L1F) :SEQ ID NO: 15 Primer (HPV18L1F):

5'- GAATTCATGTGCCTGTATACACGGGTCCTG-3' (EcoRI 절단부위 포함)
5'- GAATTC ATGTGCCTGTATACACGGGTCCTG-3 '(Including EcoR I cleavage)

서열번호 16 프라이머 (HPV18L1R) :SEQ ID NO: 16 Primer (HPV18L1R):

5'- GCATGAGTCGACCTTCCTGGCACGTACACGCAC-3' (SalI 절단부위 포함)
5'- GCATGA GTCGAC CTTCCTGGCACGTACACGCAC-3 '(includes Sal I cut)

DNA 사이즈 마커(size marker)와 함께 1% 아가로스 겔에 전기영동하여 약 1.5 kb 위치에 각각의 밴드가 존재하는 것을 확인하였다. PCR이 끝난 반응 혼합물을 1% 아가로스 겔에 전기영동하여, PCR을 통해 증폭된 약 1.5 kb의 DNA 산물을 PCR quick-spinTM PCR Product Purification Kit(Intron, Korea)을 사용하여 정제하여 회수하였다. 회수된 HPV 6, HPV 11, HPV 18 DNA를 제한효소 EcoRI 및 SalI으로, HPV 16 DNA를 제한효소 BglII 및 SalI으로 각각 절단한 다음, 유전자 절편을 각각 정제하고 회수하였다. 정제된 유전자 절편을 동일한 제한효소로 절단된 발현벡터 pET-28a-SV40에 각각 삽입하여 T4 DNA 연결효소를 사용하여 실온에서 3시간 동안 연결반응을 시켜 연결시킨 후, E. coli BL21 에 형질전환시켰다. (pET-28a-SV40/HPV16 L1을 제작하기 위하여 제한효소 BglII 와 BamHI은 annealing 서열이 동일함으로 pET-28a-SV40을 BamHI으로, HPV 16 L1을 BglII 로 절단하여 연결하였다.) 형질전환체들로부터 플라스미드 DNA를 분리한 다음, 상기의 제한효소로 절단한 후, 1% 아가로스 겔에 DNA 사이즈 마커와 함께 전기영동하여 각각의 유전자가 발현벡터 내에 정확히 삽입되어 있음을 재확인하였다. 상기와 같이 구축된 HPV 6 L1 유전자의 발현을 위해 구축된 발현벡터를 pET-28a-SV40/HPV6 L1으로(도 1), HPV 11 L1 유전자의 발현을 위해 구축된 발현벡터를 pET-28a-SV40/HPV11 L1으로(도 2), HPV 16 L1 유전자의 발현을 위해 구축된 발현벡터를 pET-28a-SV40/HPV16 L1으로(도 3) HPV 18 L1 유전자의 발현을 위해 구축된 발현벡터를 pET-28a-SV40/HPV18 L1으로 명명하였다(도 4).
Electrophoresis on 1% agarose gels with DNA size markers confirmed the presence of each band at about 1.5 kb. After the PCR reaction mixture was electrophoresed on a 1% agarose gel, a DNA product of about 1.5 kb amplified by PCR was recovered by using a PCR quick-spin TM PCR Product Purification Kit (Intron, Korea). The recovered HPV 6, HPV 11, HPV 18 DNA was digested with restriction enzymes EcoR I and Sal I, and HPV 16 DNA was digested with restriction enzymes Bgl II and Sal I, respectively, and the gene fragments were respectively purified and recovered. Purified gene fragments were inserted into expression vectors pET-28a-SV40 cut with the same restriction enzyme, and then ligated for 3 hours at room temperature using T4 DNA ligase, and then transformed into E. coli BL21. . (In order to produce pET-28a-SV40 / HPV16 L1, restriction enzymes Bgl II and Bam HI have the same annealing sequence, and thus, pET-28a-SV40 was cut into Bam HI and HPV 16 L1 was cut into Bgl II.) Plasmid DNA was isolated from the transformants, digested with the above restriction enzymes, and electrophoresed with DNA size markers on a 1% agarose gel to confirm that each gene was correctly inserted into the expression vector. The expression vector constructed for expression of the HPV 6 L1 gene constructed as described above is pET-28a-SV40 / HPV6 L1 (FIG. 1), and the expression vector constructed for expression of the HPV 11 L1 gene is pET-28a-SV40. PET-28a-SV40 / HPV16 L1 (FIG. 3) Expression vector constructed for expression of HPV 16 L1 gene (FIG. 2), pET- Named 28a-SV40 / HPV18 L1 (FIG. 4).

실시예Example 2:  2: HPVHPV 6  6 L1L1 , , HPVHPV 11  11 L1L1 , , HPVHPV 16  16 L1L1 , , HPVHPV 18  18 L1L1 포함 융합  Include fusion 폴리펩타이드의Of the polypeptide 발현과 정제 Expression and Purification

대장균에 형질전환 시킨 재조합 균주 E. coli BL21 pET-28a-SV40/HPV6 L1, E. coli BL21 pET-28a-SV40/HPV11 L1, E. coli BL21 pET-28a-SV40/HPV16 L1 및 E. coli BL21 pET-28a-SV40/HPV18 L1으로부터 N 말단에 히스티딘 택(Histidine tag)이, C말단에 SV40이 융합된 HPV 6 L1항원, HPV11 L1항원, HPV16 L1항원과 HPV18 L1항원으로 각각 발현시킨 후, mouse anti-His 항체를 일차 항체로 사용한 웨스턴 블랏을 시행하여 발현 여부를 확인하였다. 그 결과는 도 5 내지 8에 나타내었다. Recombinant strains transformed into E. coli E. coli BL21 pET-28a-SV40 / HPV6 L1, E. coli BL21 pET-28a-SV40 / HPV11 L1, E. coli BL21 pET-28a-SV40 / HPV16 L1 and E. coli BL21 The histidine tag at the N terminus and the HPV 6 L1 antigen, HPV11 L1 antigen, HPV16 L1 antigen, HPV16 L1 antigen and HPV18 L1 antigen were respectively expressed at the N-terminus of pET-28a-SV40 / HPV18 L1. Western blot using anti-His antibody as the primary antibody was performed to confirm expression. The results are shown in FIGS. 5 to 8.

상기 재조합 균주들로부터 발현된 융합 단백질은 아래와 같이 정제하였다. Fusion proteins expressed from the recombinant strains were purified as follows.

재조합 플라스미드를 가지고 있는 재조합 균주들을 100 ㎍/㎕ 암피실린이 첨가된 LB 액체 배지에 접종하여 37℃에서 16시간 동안 배양한 다음 500 ul를 100 ㎍/㎕ 암피실린이 첨가된 LB 액체 배지에 접종하여 100 ㎖에 접종하여 37℃에서 배양하였다. 600 nm에서의 흡광도가 0.4정도가 되었을 때, IPTG(isopropyl β-D-galactopyranoside)를 최종농도가 0.1 mM이 되도록 첨가하고 20℃ 48시간 배양하여 단백발현을 유도하였다. 6,000 rpm으로 15분간 원심 분리하여 균체 (1.4 g/wet weight)를 회수한 다음 CompleteTM Protease Inhibitor Cocktail(Santa Cruz Biotechnology)이 포함된 5 ml의 초음파처리 완충액(sonication buffer)(50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 5 mM EDTA)에 현탁한 다음 최종농도 0.1 ㎎/㎖ lysozyme을 첨가하고 4℃에서 1시간 lysis 시킨다. 초음파로 파쇄한 후 1% triton x-100을 첨가하고 4℃에서 1시간 반응시킨 다음 14,000 rpm으로 15분간 원심 분리하여 대장균 세포 파편(cell debris)을 제거하였다. 다음으로 상기 상층액을 Ni-NTA(Ni-Nititrilotriacetic acid) 비드와 상온에서 1시간 반응시킨 다음 용출 버퍼(20 mM Tris, pH 7.9, 500 mM NaCl, 500 mM Imidazole)를 사용하여 10분간 상온에서 반응하여 최종적으로 정제물을 회수하였다.
Recombinant strains containing the recombinant plasmids were inoculated in LB liquid medium containing 100 μg / μl ampicillin and incubated for 16 hours at 37 ° C., then 500 ul was inoculated in LB liquid medium containing 100 μg / μl ampicillin and then 100 ml. Inoculated to and incubated at 37 ℃. When the absorbance at 600 nm was about 0.4, IPTG (isopropyl β-D-galactopyranoside) was added to a final concentration of 0.1 mM and incubated at 20 ° C. for 48 hours to induce protein expression. Cells (1.4 g / wet weight) were recovered by centrifugation at 6,000 rpm for 15 minutes and then 5 ml of sonication buffer (50 mM Tris-HCl [1] containing Complete TM Protease Inhibitor Cocktail (Santa Cruz Biotechnology) [ pH 8.0], 150 mM NaCl, 5 mM EDTA), and then the final concentration of 0.1 mg / ml lysozyme is added and lysed at 4 ° C for 1 hour. After crushing by ultrasound, 1% triton x-100 was added and reacted at 4 ° C. for 1 hour, followed by centrifugation at 14,000 rpm for 15 minutes to remove E. coli cell debris. Next, the supernatant was reacted with Ni-NTA (Ni-Nititrilotriacetic acid) beads at room temperature for 1 hour, followed by reaction at room temperature for 10 minutes using an elution buffer (20 mM Tris, pH 7.9, 500 mM NaCl, 500 mM Imidazole). Finally, the purified product was recovered.

실시예Example 3: 정제된 융합  3: purified fusion 폴리펩타이드를The polypeptide 포함하는  Containing 비드Bead 어레이용  For arrays 비드의Bead 제작 making

비드 어레이용 비드를 제조하기 위해 상기 실시예 2의 방법을 통해 얻어진 정제된 융합 폴리펩타이드에 루미넥스 비드(Luminex bead)를 하기와 같은 방법으로 결합하여 제작하였다. In order to prepare beads for the bead array, Luminex beads (Luminex bead) was prepared by binding to the purified fusion polypeptide obtained through the method of Example 2 as described below.

루미넥스 비드(Luminex bead)를 5 X 106개의 농도로 준비하고, 100 ㎕의 100 mM sodium phosphate(pH 6.2) 완충용액으로 세척 후, 50 mg/mL의 N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide(EDC)와 N-hydroxysuccinimide(NHS)를 각각 10 ㎕씩 첨가하여 차광된 실온에서 20분간 비드를 활성시켰다. 활성화된 비드는 250 ㎕의 커플링 완충용액(2-morpholinoethanesulfonic acid, 50 mmol/L, pH 5.0]로 2번 세척한 후, 상기 정제된 융합 폴리펩타이드와 혼합하여 500 ㎕의 커플링 완충용액 내에서 2시간동안 반응시킨 후 커플링 완충용액을 원심분리하여 제거하고, 다시 500 ㎕의 PBS-RBN 완충용액을 첨가하여 30분간 반응시킨다. 반응 후 PBS-RBN 완충용액을 제거하고, 1 mL의 PBS-TBN 완충용액[0.1% BSA, 0.02% sodium azide, 150 mM sodium chloride, 50 mM sodium phosphate monobasic anhydrous, 0.02% tween-20; pH 7.4]으로 2회 세척하여 최종적으로 200 ㎕의 PBS-TBN 완충용액을 첨가한 후 사용하였다.
Luminex beads were prepared at a concentration of 5 × 10 6 , washed with 100 μl of 100 mM sodium phosphate (pH 6.2) buffer, and then 50 mg / mL of N- (3-dimethylaminopropyl) -N '. 10 μl of -ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added to activate beads for 20 minutes at room temperature. Activated beads were washed twice with 250 μl of coupling buffer (2-morpholinoethanesulfonic acid, 50 mmol / L, pH 5.0) and then mixed with the purified fusion polypeptide in 500 μl coupling buffer. After the reaction for 2 hours, the coupling buffer was removed by centrifugation, followed by reaction for 30 minutes by addition of 500 µl of PBS-RBN buffer, followed by removal of PBS-RBN buffer solution, and 1 mL of PBS- Wash twice with TBN buffer [0.1% BSA, 0.02% sodium azide, 150 mM sodium chloride, 50 mM sodium phosphate monobasic anhydrous, 0.02% tween-20; pH 7.4] and finally 200 μl of PBS-TBN buffer Used after addition.

실시예Example 4: 멀티플렉스  4: Multiplex 루미넥스Luminex 분석 analysis

4가 HPV 백신(서바릭스, GSK)과 Placebo 백신을 접종한 그룹을 대상으로 백신 접종 후 3개월 후에 혈액을 채취하여 혈청을 분리 한 후 개발된 HPV6 L1 항원, HPV11 L1 항원, HPV16 L1 항원 및 HPV18 L1 항원을 이용하여 실제로 혈액 내 HPV 항체의 양을 측정하였다. 융합 폴리펩타이드가 결합된 비드 혼합액을 희석된 혈청과 필터 플레이트에서 1시간 동안 반응하였다. 반응이 끝난 후 여과하여 혈청을 제거하고, 다시 여과방법을 사용하여 세척한다. 비오틴(biotin)이 결합된 2차 항체(goat anti-human IgG)를 1:1,000으로 희석하여 30분간 반응시킨다. 마지막으로 발색 시약인 스트렙타비딘-알-피코에리트린(streptavidin-R-phycoerythrin)을 첨가하여 30분간 반응시키고, Luminex analyzer로 median fluorescence intensity (MFI)를 측정하였다. Luminex analyzer는 2개의 레이저를 이용하여 한 개는 Bead 번호를 한 개는 반응된 피코에리트린의 양을 계산하여 수치로 나타낸다. 각각의 HPV 항체 유형의 판정은 HPV 항체 유형을 대표하는 비드의 MFI 값으로 대변되고, 이를 통해 HPV 항체 유형을 판정한다. 그 결과를 표 1 및 표 2에 나타내었다.
HPV6 L1 antigen, HPV11 L1 antigen, HPV16 L1 antigen, and HPV18 L1 developed after the blood was collected and serum separated 3 months after vaccination for the group of vaccinated tetravalent HPV vaccine (Surbarix, GSK) and Placebo vaccine The antigen was used to actually measure the amount of HPV antibody in the blood. The bead mixture to which the fusion polypeptide was bound was reacted for 1 hour in the diluted serum and filter plate. After completion of the reaction, the serum is removed by filtration and washed again using filtration. Biotin-conjugated secondary antibody (goat anti-human IgG) is diluted 1: 1,000 and reacted for 30 minutes. Finally, the reaction was performed for 30 minutes by the addition of streptavidin-R-phycoerythrin, a color developing reagent, and median fluorescence intensity (MFI) was measured by a Luminex analyzer. Luminex analyzer calculates the amount of picoerythrin reacted with two lasers, one bead number and one reacted. The determination of each HPV antibody type is represented by the MFI value of the beads representing the HPV antibody type, through which the HPV antibody type is determined. The results are shown in Tables 1 and 2.

Figure pat00001
Figure pat00001

표 1에 나타난 바와 같이, 총 22명 (4가 HPV 백신접종자 7명, Placebo 백신 접종자 15명)을 대상으로 실시하였으며, 4가 HPV 백신 접종자에게서만 HPV 6 및 HPV 11 타입에 대한 항체가 검출된 것을 확인할 수 있었다 (HPV6에 대한 cutoff= 500, HPV11에 대한 cutoff=1000).
As shown in Table 1, a total of 22 subjects (7 tetravalent HPV vaccinates and 15 Placebo vaccinates) were performed and antibodies to HPV 6 and HPV 11 types were detected only in the tetravalent HPV vaccinates. (Cutoff = 500 for HPV6, cutoff = 1000 for HPV11).

Figure pat00002
Figure pat00002

표 2에 나타난 바와 같이, 총 11명 (4가 HPV 백신접종자 3명, 2가 HPV 백신접종자 3명, Placebo 백신 접종자 5명)을 대상으로 실시하였으며, 4가 HPV 백신 접종자와 2가 HPV 백신접종자에게서 HPV 16 및 HPV 18 타입에 대한 항체가 검출된 것을 확인할 수 있었다 (HPV16에 대한 cutoff= 62, HPV18에 대한 cutoff=64).
As shown in Table 2, a total of 11 subjects (three tetravalent HPV vaccinates, three bivalent HPV vaccinates, and five Placebo vaccinates) were administered. It was confirmed that antibodies to HPV 16 and HPV 18 types were detected in (cutoff = 62 for HPV16, cutoff = 64 for HPV18).

<110> GYN COS CO., LTD., LTD. AHN, Woong Shick <120> Screening Kit for Human Papillomavirus antibody using fusion polypeptide HPV antigen <130> PA120395KR <160> 16 <170> KopatentIn 1.71 <210> 1 <211> 553 <212> PRT <213> Artificial Sequence <220> <223> fusion polypeptide HPV 6 L1 <400> 1 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Glu Phe Met Trp Arg Pro Ser Asp Ser Thr Val Tyr Val Pro 35 40 45 Pro Pro Asn Pro Val Ser Lys Val Val Ala Thr Asp Ala Tyr Val Thr 50 55 60 Arg Thr Asn Ile Phe Tyr His Ala Ser Ser Ser Arg Leu Leu Ala Val 65 70 75 80 Gly His Pro Tyr Phe Ser Ile Lys Arg Ala Asn Lys Thr Val Val Pro 85 90 95 Lys Val Ser Gly Tyr Gln Tyr Arg Val Phe Lys Val Val Leu Pro Asp 100 105 110 Pro Asn Lys Phe Ala Leu Pro Asp Ser Ser Leu Phe Asp Pro Thr Thr 115 120 125 Gln Arg Leu Val Trp Ala Cys Thr Gly Leu Glu Val Gly Arg Gly Gln 130 135 140 Pro Leu Gly Val Gly Val Ser Gly His Pro Phe Leu Asn Lys Tyr Asp 145 150 155 160 Asp Val Glu Asn Ser Gly Ser Gly Gly Asn Pro Gly Gln Asp Asn Arg 165 170 175 Val Asn Val Gly Met Asp Tyr Lys Gln Thr Gln Leu Cys Met Val Gly 180 185 190 Cys Ala Pro Pro Leu Gly Glu His Trp Gly Lys Gly Lys Gln Cys Thr 195 200 205 Asn Thr Pro Val Gln Ala Gly Asp Cys Pro Pro Leu Glu Leu Ile Thr 210 215 220 Ser Val Ile Gln Asp Gly Asp Met Val Asp Thr Gly Phe Gly Ala Met 225 230 235 240 Asn Phe Ala Asp Leu Gln Thr Asn Lys Ser Asp Val Pro Ile Asp Ile 245 250 255 Cys Gly Thr Thr Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ala Ala Asp 260 265 270 Pro Tyr Gly Asp Arg Leu Phe Phe Phe Leu Arg Lys Glu Gln Met Phe 275 280 285 Ala Arg His Phe Phe Asn Arg Ala Gly Glu Val Gly Glu Pro Val Pro 290 295 300 Asp Thr Leu Ile Ile Lys Gly Ser Gly Asn Arg Thr Ser Val Gly Ser 305 310 315 320 Ser Ile Tyr Val Asn Thr Pro Ser Gly Ser Leu Val Ser Ser Glu Ala 325 330 335 Gln Leu Phe Asn Lys Pro Tyr Trp Leu Gln Lys Ala Gln Gly His Asn 340 345 350 Asn Gly Ile Cys Trp Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr 355 360 365 Thr Arg Ser Thr Asn Met Thr Leu Cys Ala Ser Val Thr Thr Ser Ser 370 375 380 Thr Tyr Thr Asn Ser Asp Tyr Lys Glu Tyr Met Arg His Val Glu Glu 385 390 395 400 Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Ser Ile Thr Leu Ser Ala 405 410 415 Glu Val Met Ala Tyr Ile His Thr Met Asn Pro Ser Val Leu Glu Asp 420 425 430 Trp Asn Phe Gly Leu Ser Pro Pro Pro Asn Gly Thr Leu Glu Asp Thr 435 440 445 Tyr Arg Tyr Val Gln Ser Gln Ala Ile Thr Cys Gln Lys Pro Thr Pro 450 455 460 Glu Lys Glu Lys Pro Asp Pro Tyr Lys Asn Leu Ser Phe Trp Glu Val 465 470 475 480 Asn Leu Lys Glu Lys Phe Ser Ser Glu Leu Asp Gln Tyr Pro Leu Gly 485 490 495 Arg Lys Phe Leu Leu Gln Ser Gly Tyr Arg Gly Arg Ser Ser Ile Arg 500 505 510 Thr Gly Val Lys Arg Pro Ala Val Ser Lys Ala Ser Ala Ala Pro Lys 515 520 525 Arg Lys Arg Ala Lys Thr Lys Arg Val Asp Lys Leu Ala Asn Leu Pro 530 535 540 His Leu Pro Leu Asn Leu Lys His Xaa 545 550 <210> 2 <211> 554 <212> PRT <213> Artificial Sequence <220> <223> fusion polypeptide HPV 11 L1 <400> 2 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Glu Phe Met Trp Arg Pro Ser Asp Ser Thr Val Tyr Val Pro 35 40 45 Pro Pro Asn Pro Val Ser Lys Val Val Ala Thr Asp Ala Tyr Val Lys 50 55 60 Arg Thr Asn Ile Phe Tyr His Ala Ser Ser Ser Arg Leu Leu Ala Val 65 70 75 80 Gly His Pro Tyr Tyr Ser Ile Lys Lys Val Asn Lys Thr Val Val Pro 85 90 95 Lys Val Ser Gly Tyr Gln Tyr Arg Val Phe Lys Val Val Leu Pro Asp 100 105 110 Pro Asn Lys Phe Ala Leu Pro Asp Ser Ser Leu Phe Asp Pro Thr Thr 115 120 125 Gln Arg Leu Val Trp Ala Cys Thr Gly Leu Glu Val Gly Arg Gly Gln 130 135 140 Pro Leu Gly Val Gly Val Ser Gly His Pro Leu Leu Asn Lys Tyr Asp 145 150 155 160 Asp Val Glu Asn Ser Gly Gly Tyr Gly Gly Asn Pro Gly Gln Asp Asn 165 170 175 Arg Val Asn Val Gly Met Asp Tyr Lys Gln Thr Gln Leu Cys Met Val 180 185 190 Gly Cys Ala Pro Pro Leu Gly Glu His Trp Gly Lys Gly Thr Gln Cys 195 200 205 Ser Asn Thr Ser Val Gln Asn Gly Asp Cys Pro Pro Leu Glu Leu Ile 210 215 220 Thr Ser Val Ile Gln Asp Gly Asp Met Val Asp Thr Gly Phe Gly Ala 225 230 235 240 Met Asn Phe Ala Asp Leu Gln Thr Asn Lys Ser Asp Val Pro Leu Asp 245 250 255 Ile Cys Gly Thr Val Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ala Ala 260 265 270 Asp Pro Tyr Gly Asp Arg Leu Phe Phe Tyr Leu Arg Lys Glu Gln Met 275 280 285 Phe Ala Arg His Phe Phe Asn Arg Ala Gly Thr Val Gly Glu Pro Val 290 295 300 Pro Asp Asp Leu Leu Val Lys Gly Gly Asn Asn Arg Ser Ser Val Ala 305 310 315 320 Ser Ser Ile Tyr Val His Thr Pro Ser Gly Ser Leu Val Ser Ser Glu 325 330 335 Ala Gln Leu Phe Asn Lys Pro Tyr Trp Leu Gln Lys Ala Gln Gly His 340 345 350 Asn Asn Gly Ile Cys Trp Gly Asn His Leu Phe Val Thr Val Val Asp 355 360 365 Thr Thr Arg Ser Thr Asn Met Thr Leu Cys Ala Ser Val Ser Lys Ser 370 375 380 Ala Thr Tyr Thr Asn Ser Asp Tyr Lys Glu Tyr Met Arg His Val Glu 385 390 395 400 Glu Phe Asp Leu Gln Phe Ile Phe Gln Leu Cys Ser Ile Thr Leu Ser 405 410 415 Ala Glu Val Met Ala Tyr Ile His Thr Met Asn Pro Ser Val Leu Glu 420 425 430 Asp Trp Asn Phe Gly Leu Ser Pro Pro Pro Asn Gly Thr Leu Glu Asp 435 440 445 Thr Tyr Arg Tyr Val Gln Ser Gln Ala Ile Thr Cys Gln Lys Pro Thr 450 455 460 Pro Glu Lys Glu Lys Gln Asp Pro Tyr Lys Asp Met Ser Phe Trp Glu 465 470 475 480 Val Asn Leu Lys Glu Lys Phe Ser Ser Glu Leu Asp Gln Phe Pro Leu 485 490 495 Gly Arg Lys Phe Leu Leu Gln Ser Gly Tyr Arg Gly Arg Thr Ser Ala 500 505 510 Arg Thr Gly Ile Lys Arg Pro Ala Val Ser Lys Pro Ser Thr Ala Pro 515 520 525 Lys Arg Lys Arg Thr Lys Thr Lys Lys Val Asp Lys Leu Ala Asn Leu 530 535 540 Pro His Leu Pro Leu Asn Leu Lys His Xaa 545 550 <210> 3 <211> 549 <212> PRT <213> Artificial Sequence <220> <223> fusion polypeptide HPV 16 L1 <400> 3 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Glu Phe Glu Leu Arg Arg Gln Val Tyr Leu Pro Pro Val Pro 35 40 45 Val Ser Lys Val Val Ser Thr Asp Glu Tyr Val Ala Arg Thr Asn Ile 50 55 60 Tyr Tyr His Ala Gly Thr Ser Arg Leu Leu Ala Val Gly His Pro Tyr 65 70 75 80 Phe Pro Ile Lys Lys Pro Asn Asn Asn Lys Ile Leu Val Pro Lys Val 85 90 95 Ser Gly Leu Gln Tyr Arg Val Phe Arg Ile His Leu Pro Asp Pro Asn 100 105 110 Lys Phe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr Gln Arg 115 120 125 Leu Val Trp Ala Cys Val Gly Val Glu Val Gly Arg Gly Gln Pro Leu 130 135 140 Gly Val Gly Ile Ser Gly His Pro Leu Leu Asn Lys Leu Asp Asp Thr 145 150 155 160 Glu Asn Ala Ser Ala Tyr Ala Ala Asn Ala Gly Val Asp Asn Arg Glu 165 170 175 Cys Ile Ser Met Asp Tyr Lys Gln Thr Gln Leu Cys Leu Ile Gly Cys 180 185 190 Lys Pro Pro Ile Gly Glu His Trp Gly Lys Gly Ser Pro Cys Thr Asn 195 200 205 Val Ala Val Asn Pro Gly Asp Cys Pro Pro Leu Glu Leu Ile Asn Thr 210 215 220 Val Ile Gln Asp Gly Asp Met Val Asp Thr Gly Phe Gly Ala Met Asp 225 230 235 240 Phe Thr Thr Leu Gln Ala Asn Lys Ser Glu Val Pro Leu Asp Ile Cys 245 250 255 Thr Ser Ile Cys Lys Tyr Pro Asp Tyr Ile Lys Met Val Ser Glu Pro 260 265 270 Tyr Gly Asp Ser Leu Phe Phe Tyr Leu Arg Arg Glu Gln Met Phe Val 275 280 285 Arg His Leu Phe Asn Arg Ala Gly Ala Val Gly Glu Asn Val Pro Asp 290 295 300 Asp Leu Tyr Ile Lys Gly Ser Gly Ser Thr Ala Asn Leu Ala Ser Ser 305 310 315 320 Asn Tyr Phe Pro Thr Pro Ser Gly Ser Met Val Thr Ser Asp Ala Gln 325 330 335 Ile Phe Asn Lys Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn Asn 340 345 350 Gly Ile Cys Trp Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr Thr 355 360 365 Arg Ser Thr Asn Met Ser Leu Cys Ala Ala Ile Ser Thr Ser Glu Thr 370 375 380 Thr Tyr Lys Asn Thr Asn Phe Lys Glu Tyr Leu Arg His Gly Glu Glu 385 390 395 400 Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Lys Ile Thr Leu Thr Ala 405 410 415 Asp Val Met Thr Tyr Ile His Ser Met Asn Ser Thr Ile Leu Glu Asp 420 425 430 Trp Asn Phe Gly Leu Gln Pro Pro Pro Gly Gly Thr Leu Glu Asp Thr 435 440 445 Tyr Arg Phe Val Thr Ser Gln Ala Ile Ala Cys Gln Lys His Thr Pro 450 455 460 Pro Ala Pro Lys Glu Asp Pro Leu Lys Lys Tyr Thr Phe Trp Glu Val 465 470 475 480 Asn Leu Lys Glu Lys Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly 485 490 495 Arg Lys Phe Leu Leu Gln Ala Gly Leu Lys Ala Lys Pro Lys Phe Thr 500 505 510 Leu Gly Lys Arg Lys Ala Thr Pro Thr Thr Ser Ser Thr Ser Thr Thr 515 520 525 Ala Lys Arg Lys Lys Arg Lys Leu Lys Leu Lys Pro Pro Thr Pro Pro 530 535 540 Pro Glu Pro Glu Thr 545 <210> 4 <211> 550 <212> PRT <213> Artificial Sequence <220> <223> fusion polypeptide HPV 18 L1 <400> 4 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Glu Phe Val Tyr Leu Pro Pro Pro Ser Val Ala Arg Val Val 35 40 45 Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser Ile Phe Tyr His Ala Gly 50 55 60 Ser Ser Arg Leu Leu Thr Val Gly Asn Pro Tyr Phe Arg Val Pro Ala 65 70 75 80 Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys Val Ser Ala Tyr Gln Tyr 85 90 95 Arg Val Phe Arg Val Gln Leu Pro Asp Pro Asn Lys Phe Gly Leu Pro 100 105 110 Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln Arg Leu Val Trp Ala Cys 115 120 125 Ala Gly Val Glu Ile Gly Arg Gly Gln Pro Leu Gly Val Gly Leu Ser 130 135 140 Gly His Pro Phe Tyr Asn Lys Leu Asp Asp Thr Glu Ser Ser His Ala 145 150 155 160 Ala Thr Ser Asn Val Ser Glu Asp Val Arg Asp Asn Val Ser Val Asp 165 170 175 Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly Cys Ala Pro Ala Ile Gly 180 185 190 Glu His Trp Ala Lys Gly Thr Ala Cys Lys Ser Arg Pro Leu Ser Gln 195 200 205 Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn Thr Val Leu Glu Asp Gly 210 215 220 Asp Met Val Asp Thr Gly Tyr Gly Ala Met Asp Phe Ser Thr Leu Gln 225 230 235 240 Asp Thr Lys Cys Glu Val Pro Leu Asp Ile Cys Gln Ser Ile Cys Lys 245 250 255 Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp Pro Tyr Gly Asp Ser Met 260 265 270 Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe Ala Arg His Phe Trp Asn 275 280 285 Arg Ala Gly Thr Met Gly Asp Thr Val Pro Gln Ser Leu Tyr Ile Lys 290 295 300 Gly Thr Gly Met Arg Ala Ser Pro Gly Ser Cys Val Tyr Ser Pro Ser 305 310 315 320 Pro Ser Gly Ser Ile Val Thr Ser Asp Ser Gln Leu Phe Asn Lys Pro 325 330 335 Tyr Trp Leu His Lys Ala Gln Gly His Asn Asn Gly Val Cys Trp His 340 345 350 Asn Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser Thr Asn Leu 355 360 365 Thr Ile Cys Ala Ser Thr Gln Ser Pro Val Pro Gly Gln Tyr Asp Ala 370 375 380 Thr Lys Phe Lys Gln Tyr Ser Arg His Val Glu Glu Tyr Asp Leu Gln 385 390 395 400 Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu Thr Ala Asp Val Met Ser 405 410 415 Tyr Ile His Ser Met Asn Ser Ser Ile Leu Glu Asp Trp Asn Phe Gly 420 425 430 Val Pro Pro Pro Pro Thr Thr Ser Leu Val Asp Thr Tyr Arg Phe Val 435 440 445 Gln Ser Val Ala Ile Thr Cys Gln Lys Asp Ala Ala Pro Ala Glu Asn 450 455 460 Lys Asp Pro Tyr Asp Lys Leu Lys Phe Trp Asn Val Asp Leu Lys Glu 465 470 475 480 Lys Phe Ser Leu Asp Leu Asp Gln Tyr Pro Leu Gly Arg Lys Phe Leu 485 490 495 Val Gln Ala Gly Leu Arg Arg Lys Pro Thr Ile Gly Pro Arg Lys Arg 500 505 510 Ser Ala Pro Ser Ala Thr Thr Ser Ser Lys Pro Ala Lys Arg Val Arg 515 520 525 Val Arg Ala Arg Lys Val Asp Lys Leu Ala Asn Leu Pro His Leu Pro 530 535 540 Leu Asn Leu Lys His Xaa 545 550 <210> 5 <211> 1658 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding fusion polypeptide HPV 6 L1 <400> 5 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcat gtggcggcct 120 agcgacagca cagtatatgt gcctcctcct aaccctgtat ccaaagttgt tgccacggat 180 gcttatgtta ctcgcaccaa catattttat catgccagca gttctagact tcttgcagtg 240 ggacatcctt atttttccat aaaacgggct aacaaaactg ttgtgccaaa ggtgtcagga 300 tatcaataca gggtatttaa ggtggtgtta ccagatccta acaaatttgc attgcctgac 360 tcgtctcttt tcgatcccac aacacaacgt ttagtatggg catgcacagg cctagaggtg 420 ggcaggggac agccattagg tgtgggtgta agtggacatc ctttcctaaa taaatatgat 480 gatgttgaaa attcagggag tggtggtaac cctggacagg ataacagggt taatgtaggt 540 atggattata aacaaacaca attatgcatg gttggatgtg cccccccttt gggcgagcat 600 tggggtaaag gtaaacagtg tactaataca cctgtacagg ctggtgactg cccgccctta 660 gaacttatta ccagtgttat acaggatggc gatatggttg acacaggctt tggtgctatg 720 aattttgctg atttgcagac caataaatca gatgttccta ttgacatatg tggcactaca 780 tgtaaatatc cagattattt acaaatggct gcagacccat atggtgatag attatttttt 840 tttctacgga aggaacaaat gtttgccaga cattttttta acagggctgg cgaggtgggg 900 gaacctgtgc ctgatacact tataattaag ggtagtggaa atcgcacgtc tgtagggagt 960 agtatatatg ttaacacccc gagcggctct ttggtgtcct ctgaggcaca attgtttaat 1020 aagccatatt ggctacaaaa agcccaggga cataacaatg gtatttgttg gggtaatcaa 1080 ctgtttgtta ctgtggtaga taccacacgc agtaccaaca tgacattatg tgcatccgta 1140 actacatctt ccacatacac caattctgat tataaagagt acatgcgtca tgtggaagag 1200 tatgatttac aatttatttt tcaattatgt agcattacat tgtctgctga agtaatggcc 1260 tatattcaca caatgaatcc ctctgttttg gaagactgga actttgggtt atcgcctccc 1320 ccaaatggta cattagaaga tacctatagg tatgtgcagt cacaggccat tacctgtcaa 1380 aagcccactc ctgaaaagga aaagccagat ccctataaga accttagttt ttgggaggtt 1440 aatttaaaag aaaagttttc tagtgaattg gatcagtatc ctttgggacg caagtttttg 1500 ttacaaagtg gatatagggg acggtcctct attcgtacag gtgttaagcg ccctgctgtt 1560 tccaaagcct ctgctgcccc taaacgtaag cgcgccaaaa ctaaaagggt cgacaagctt 1620 gcaaacctcc cacacctccc cctgaacctg aaacataa 1658 <210> 6 <211> 1661 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding fusion polypeptide HPV 11 L1 <400> 6 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcat gtggcggcct 120 agcgacagca cagtatatgt gcctcctccc aaccctgtat ccaaggttgt tgccacggat 180 gcgtatgtta aacgcaccaa catattttat catgccagca gttctagact ccttgctgtg 240 ggacatccat attactctat caaaaaagtt aacaaaacag ttgtaccaaa ggtgtctgga 300 tatcaatata gagtgtttaa ggtagtgttg ccagatccta acaagtttgc attacctgat 360 tcatccctgt ttgaccccac tacacagcgt ttagtatggg cgtgcacagg gttggaggta 420 ggcaggggtc aacctttagg cgttggtgtt agtgggcatc cattgctaaa caaatatgat 480 gatgtagaaa atagtggtgg gtatggtggt aatcctggtc aggataatag ggttaatgta 540 ggtatggatt ataaacaaac ccagctatgt atggtgggct gtgctccacc gttaggtgaa 600 cattggggta agggtacaca atgttcaaat acctctgtac aaaatggtga ctgccccccg 660 ttggaactta ttaccagtgt tatacaggat ggggacatgg ttgatacagg ctttggtgct 720 atgaattttg cagacttaca aaccaataaa tcggatgttc cccttgatat ttgtggaact 780 gtctgcaaat atcctgatta tttgcaaatg gctgcagacc cttatggtga taggttgttt 840 ttttatttgc gaaaggaaca aatgtttgct agacactttt ttaatagggc cggtactgtg 900 ggggaacctg tgcctgatga cctgttggta aaagggggta ataacagatc atctgtagct 960 agtagtattt atgtacatac acctagtggc tcattggtgt cttcagaggc tcaattattt 1020 aataaaccat attggcttca aaaggctcag ggacataaca atggtatttg ctggggaaac 1080 cacttgtttg ttactgtggt agataccaca cgcagtacaa atatgacact atgtgcatct 1140 gtgtctaaat ctgctacata cactaattca gattataagg aatacatgcg ccatgtggag 1200 gagtttgatt tacagtttat ttttcaattg tgtagcatta cattatctgc agaagtcatg 1260 gcctatatac acacaatgaa tccttctgtt ttggaggact ggaactttgg tttatcgcct 1320 ccaccaaatg gtacactgga ggatacttat agatatgtac agtcacaggc cattacctgt 1380 cagaaaccca cacctgaaaa agaaaaacag gatccctata aggatatgag tttttgggag 1440 gttaacttaa aagaaaagtt ttcaagtgaa ttagatcagt ttccccttgg acgtaagttt 1500 ttattgcaaa gtggatatcg aggacggacg tctgctcgta caggtataaa gcgcccagct 1560 gtgtctaagc cctctacagc ccccaaacga aaacgtacca aaaccaaaaa ggtcgacaag 1620 cttgcaaacc tcccacacct ccccctgaac ctgaaacata a 1661 <210> 7 <211> 1650 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding fusion polypeptide HPV 16 L1 <400> 7 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcga gctccgtcga 120 caagtctact tgcctcctgt cccagtatct aaggttgtaa gcacggatga atatgttgca 180 cgcacaaaca tatattatca tgcaggaaca tccagactac ttgcagttgg acatccctat 240 tttcctatta aaaaacctaa caataacaaa atattagttc ctaaagtatc aggattacaa 300 tacagggtat ttagaataca tttacctgac cccaataagt ttggttttcc tgacacctca 360 ttttataatc cagatacaca gcggctggtt tgggcctgtg taggtgttga ggtaggtcgt 420 ggtcagccat taggtgtggg cattagtggc catcctttat taaataaatt ggatgacaca 480 gaaaatgcta gtgcttatgc agcaaatgca ggtgtggata atagagaatg tatatctatg 540 gattacaaac aaacacaatt gtgtttaatt ggttgcaaac cacctatagg ggaacactgg 600 ggcaaaggat ccccatgtac caatgttgca gtaaatccag gtgattgtcc accattagag 660 ttaataaaca cagttattca ggatggtgat atggttgata ctggctttgg tgctatggac 720 tttactacat tacaggctaa caaaagtgaa gttccactgg atatttgtac atctatttgc 780 aaatatccag attatattaa aatggtgtca gaaccatatg gcgacagctt atttttttat 840 ttacgaaggg aacaaatgtt tgttagacat ttatttaata gggctggtgc tgttggtgaa 900 aatgtaccag acgatttata cattaaaggc tctgggtcta ctgcaaattt agccagttca 960 aattattttc ctacacctag tggttctatg gttacctctg atgcccaaat attcaataaa 1020 ccttattggt tacaacgagc acagggccac aataatggca tttgttgggg taaccaacta 1080 tttgttactg ttgttgatac tacacgcagt acaaatatgt cattatgtgc tgccatatct 1140 acttcagaaa ctacatataa aaatactaac tttaaggagt acctacgaca tggggaggaa 1200 tatgatttac agtttatttt tcaactgtgc aaaataacct taactgcaga cgttatgaca 1260 tacatacatt ctatgaattc cactattttg gaggactgga attttggtct acaacctccc 1320 ccaggaggca cactagaaga tacttatagg tttgtaacat cccaggcaat tgcttgtcaa 1380 aaacatacac ctccagcacc taaagaagat ccccttaaaa aatacacttt ttgggaagta 1440 aatttaaagg aaaagttttc tgcagaccta gatcagtttc ctttaggacg caaattttta 1500 ctacaagcag gattgaaggc caaaccaaaa tttacattag gaaaacgaaa agctacaccc 1560 accacctcat ctacctctac aactgctaaa cgcaaaaaac gtaagctgaa gcttaaacct 1620 cccacacctc cccctgaacc tgaaacataa 1650 <210> 8 <211> 1649 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding fusion polypeptide HPV 18 L1 <400> 8 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcgt atatcttcca 120 cctccttctg tggcaagagt tgtaaatacc gatgattatg tgactcgcac aagcatattt 180 tatcatgctg gcagctctag attattaact gttggtaatc catattttag ggttcctgca 240 ggtggtggca ataagcagga tattcctaag gtttctgcat accaatatag agtatttagg 300 gtgcagttac ctgacccaaa taaatttggt ttacctgata ctagtattta taatcctgaa 360 acacaacgtt tagtgtgggc ctgtgctgga gtggaaattg gccgtggtca gcctttaggt 420 gttggcctta gtgggcatcc attttataat aaattagatg acactgaaag ttcccatgcc 480 gccacgtcta atgtttctga ggacgttagg gacaatgtgt ctgtagatta taagcagaca 540 cagttatgta ttttgggctg tgcccctgct attggggaac actgggctaa aggcactgct 600 tgtaaatcgc gtcctttatc acagggcgat tgcccccctt tagaacttaa aaacacagtt 660 ttggaagatg gtgatatggt agatactgga tatggtgcca tggactttag tacattgcaa 720 gatactaaat gtgaggtacc attggatatt tgtcagtcta tttgtaaata tcctgattat 780 ttacaaatgt ctgcagatcc ttatggggat tccatgtttt tttgcttacg gcgtgagcag 840 ctttttgcta ggcatttttg gaatagagca ggtactatgg gtgacactgt gcctcaatcc 900 ttatatatta aaggcacagg tatgcgtgct tcacctggca gctgtgtgta ttctccctct 960 ccaagtggct ctattgttac ctctgactcc cagttgttta ataaaccata ttggttacat 1020 aaggcacagg gtcataacaa tggtgtttgc tggcataatc aattatttgt tactgtggta 1080 gataccactc gcagtaccaa tttaacaata tgtgcttcta cacagtctcc tgtacctggg 1140 caatatgatg ctaccaaatt taagcagtat agcagacatg ttgaggaata tgatttgcag 1200 tttatttttc agttgtgtac tattacttta actgcagatg ttatgtccta tattcatagt 1260 atgaatagca gtattttaga ggattggaac tttggtgttc cccccccgcc aactactagt 1320 ttggtggata catatcgttt tgtacaatct gttgctatta cctgtcaaaa ggatgctgca 1380 ccggctgaaa ataaggatcc ctatgataag ttaaagtttt ggaatgtgga tttaaaggaa 1440 aagttttctt tagacttaga tcaatatccc cttggacgta aatttttggt tcaggctgga 1500 ttgcgtcgca agcccaccat aggccctcgc aaacgttctg ctccatctgc cactacgtct 1560 tctaaacctg ccaagcgtgt gcgtgtacgt gccaggaagg tcgacaagct tgcaaacctc 1620 ccacacctcc ccctgaacct gaaacataa 1649 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer HPV6L1F <400> 9 gaattcatgt ggcggcctag cgacagc 27 <210> 10 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer HPV6L1R <400> 10 gcatgagtcg acccttttag ttttggcgcg c 31 <210> 11 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer HPV11L1F <400> 11 gaattcatgt ggcggcctag cgacagc 27 <210> 12 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer HPV11L1R <400> 12 gcatgagtcg acctttttgg ttttggtacg ttt 33 <210> 13 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer HPV16L1F <400> 13 gtcgaccgat gcaggtgact tttatttaca tc 32 <210> 14 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer HPV16L1R <400> 14 aagcttcagc ttacgttttt tgcgtttagc 30 <210> 15 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer HPV18L1F <400> 15 gaattcatgt gcctgtatac acgggtcctg 30 <210> 16 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer HPV18L1R <400> 16 gcatgagtcg accttcctgg cacgtacacg cac 33 <110> GYN COS CO., LTD., LTD.          AHN, Woong Shick <120> Screening Kit for Human Papillomavirus antibody using fusion          polypeptide HPV antigen <130> PA120395KR <160> 16 <170> Kopatentin 1.71 <210> 1 <211> 553 <212> PRT <213> Artificial Sequence <220> 223 fusion polypeptide HPV 6 L1 <400> 1 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro   1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg              20 25 30 Gly Ser Glu Phe Met Trp Arg Pro Ser Asp Ser Thr Val Tyr Val Pro          35 40 45 Pro Pro Asn Pro Val Ser Lys Val Val Ala Thr Asp Ala Tyr Val Thr      50 55 60 Arg Thr Asn Ile Phe Tyr His Ala Ser Ser Ser Arg Leu Leu Ala Val  65 70 75 80 Gly His Pro Tyr Phe Ser Ile Lys Arg Ala Asn Lys Thr Val Val Pro                  85 90 95 Lys Val Ser Gly Tyr Gln Tyr Arg Val Phe Lys Val Val Leu Pro Asp             100 105 110 Pro Asn Lys Phe Ala Leu Pro Asp Ser Ser Leu Phe Asp Pro Thr Thr         115 120 125 Gln Arg Leu Val Trp Ala Cys Thr Gly Leu Glu Val Gly Arg Gly Gln     130 135 140 Pro Leu Gly Val Gly Val Ser Gly His Pro Phe Leu Asn Lys Tyr Asp 145 150 155 160 Asp Val Glu Asn Ser Gly Ser Gly Gly Asn Pro Gly Gln Asp Asn Arg                 165 170 175 Val Asn Val Gly Met Asp Tyr Lys Gln Thr Gln Leu Cys Met Val Gly             180 185 190 Cys Ala Pro Pro Leu Gly Glu His Trp Gly Lys Gly Lys Gln Cys Thr         195 200 205 Asn Thr Pro Val Gln Ala Gly Asp Cys Pro Pro Leu Glu Leu Ile Thr     210 215 220 Ser Val Ile Gln Asp Gly Asp Met Val Asp Thr Gly Phe Gly Ala Met 225 230 235 240 Asn Phe Ala Asp Leu Gln Thr Asn Lys Ser Asp Val Pro Ile Asp Ile                 245 250 255 Cys Gly Thr Thr Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ala Ala Asp             260 265 270 Pro Tyr Gly Asp Arg Leu Phe Phe Phe Leu Arg Lys Glu Gln Met Phe         275 280 285 Ala Arg His Phe Phe Asn Arg Ala Gly Glu Val Gly Glu Pro Val Pro     290 295 300 Asp Thr Leu Ile Ile Lys Gly Ser Gly Asn Arg Thr Ser Val Gly Ser 305 310 315 320 Ser Ile Tyr Val Asn Thr Pro Ser Gly Ser Leu Val Ser Ser Glu Ala                 325 330 335 Gln Leu Phe Asn Lys Pro Tyr Trp Leu Gln Lys Ala Gln Gly His Asn             340 345 350 Asn Gly Ile Cys Trp Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr         355 360 365 Thr Arg Ser Thr Asn Met Thr Leu Cys Ala Ser Val Thr Thr Ser Ser     370 375 380 Thr Tyr Thr Asn Ser Asp Tyr Lys Glu Tyr Met Arg His Val Glu Glu 385 390 395 400 Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Ser Ile Thr Leu Ser Ala                 405 410 415 Glu Val Met Ala Tyr Ile His Thr Met Asn Pro Ser Val Leu Glu Asp             420 425 430 Trp Asn Phe Gly Leu Ser Pro Pro Pro Asn Gly Thr Leu Glu Asp Thr         435 440 445 Tyr Arg Tyr Val Gln Ser Gln Ala Ile Thr Cys Gln Lys Pro Thr Pro     450 455 460 Glu Lys Glu Lys Pro Asp Pro Tyr Lys Asn Leu Ser Phe Trp Glu Val 465 470 475 480 Asn Leu Lys Glu Lys Phe Ser Ser Glu Leu Asp Gln Tyr Pro Leu Gly                 485 490 495 Arg Lys Phe Leu Leu Gln Ser Gly Tyr Arg Gly Arg Ser Ser Ile Arg             500 505 510 Thr Gly Val Lys Arg Pro Ala Val Ser Lys Ala Ser Ala Ala Pro Lys         515 520 525 Arg Lys Arg Ala Lys Thr Lys Arg Val Asp Lys Leu Ala Asn Leu Pro     530 535 540 His Leu Pro Leu Asn Leu Lys His Xaa 545 550 <210> 2 <211> 554 <212> PRT <213> Artificial Sequence <220> <223> fusion polypeptide HPV 11 L1 <400> 2 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro   1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg              20 25 30 Gly Ser Glu Phe Met Trp Arg Pro Ser Asp Ser Thr Val Tyr Val Pro          35 40 45 Pro Pro Asn Pro Val Ser Lys Val Val Ala Thr Asp Ala Tyr Val Lys      50 55 60 Arg Thr Asn Ile Phe Tyr His Ala Ser Ser Ser Arg Leu Leu Ala Val  65 70 75 80 Gly His Pro Tyr Tyr Ser Ile Lys Lys Val Asn Lys Thr Val Val Pro                  85 90 95 Lys Val Ser Gly Tyr Gln Tyr Arg Val Phe Lys Val Val Leu Pro Asp             100 105 110 Pro Asn Lys Phe Ala Leu Pro Asp Ser Ser Leu Phe Asp Pro Thr Thr         115 120 125 Gln Arg Leu Val Trp Ala Cys Thr Gly Leu Glu Val Gly Arg Gly Gln     130 135 140 Pro Leu Gly Val Gly Val Ser Gly His Pro Leu Leu Asn Lys Tyr Asp 145 150 155 160 Asp Val Glu Asn Ser Gly Gly Tyr Gly Gly Asn Pro Gly Gln Asp Asn                 165 170 175 Arg Val Asn Val Gly Met Asp Tyr Lys Gln Thr Gln Leu Cys Met Val             180 185 190 Gly Cys Ala Pro Pro Leu Gly Glu His Trp Gly Lys Gly Thr Gln Cys         195 200 205 Ser Asn Thr Ser Val Gln Asn Gly Asp Cys Pro Pro Leu Glu Leu Ile     210 215 220 Thr Ser Val Ile Gln Asp Gly Asp Met Val Asp Thr Gly Phe Gly Ala 225 230 235 240 Met Asn Phe Ala Asp Leu Gln Thr Asn Lys Ser Asp Val Pro Leu Asp                 245 250 255 Ile Cys Gly Thr Val Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ala Ala             260 265 270 Asp Pro Tyr Gly Asp Arg Leu Phe Phe Tyr Leu Arg Lys Glu Gln Met         275 280 285 Phe Ala Arg His Phe Phe Asn Arg Ala Gly Thr Val Gly Glu Pro Val     290 295 300 Pro Asp Asp Leu Leu Val Lys Gly Gly Asn Asn Arg Ser Ser Val Ala 305 310 315 320 Ser Ser Ile Tyr Val His Thr Pro Ser Gly Ser Leu Val Ser Ser Glu                 325 330 335 Ala Gln Leu Phe Asn Lys Pro Tyr Trp Leu Gln Lys Ala Gln Gly His             340 345 350 Asn Asn Gly Ile Cys Trp Gly Asn His Leu Phe Val Thr Val Val Asp         355 360 365 Thr Thr Arg Ser Thr Asn Met Thr Leu Cys Ala Ser Val Ser Lys Ser     370 375 380 Ala Thr Tyr Thr Asn Ser Asp Tyr Lys Glu Tyr Met Arg His Val Glu 385 390 395 400 Glu Phe Asp Leu Gln Phe Ile Phe Gln Leu Cys Ser Ile Thr Leu Ser                 405 410 415 Ala Glu Val Met Ala Tyr Ile His Thr Met Asn Pro Ser Val Leu Glu             420 425 430 Asp Trp Asn Phe Gly Leu Ser Pro Pro Pro Asn Gly Thr Leu Glu Asp         435 440 445 Thr Tyr Arg Tyr Val Gln Ser Gln Ala Ile Thr Cys Gln Lys Pro Thr     450 455 460 Pro Glu Lys Glu Lys Gln Asp Pro Tyr Lys Asp Met Ser Phe Trp Glu 465 470 475 480 Val Asn Leu Lys Glu Lys Phe Ser Ser Glu Leu Asp Gln Phe Pro Leu                 485 490 495 Gly Arg Lys Phe Leu Leu Gln Ser Gly Tyr Arg Gly Arg Thr Ser Ala             500 505 510 Arg Thr Gly Ile Lys Arg Pro Ala Val Ser Lys Pro Ser Thr Ala Pro         515 520 525 Lys Arg Lys Arg Thr Lys Thr Lys Lys Val Asp Lys Leu Ala Asn Leu     530 535 540 Pro His Leu Pro Leu Asn Leu Lys His Xaa 545 550 <210> 3 <211> 549 <212> PRT <213> Artificial Sequence <220> 223 fusion polypeptide HPV 16 L1 <400> 3 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro   1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg              20 25 30 Gly Ser Glu Phe Glu Leu Arg Arg Gln Val Tyr Leu Pro Pro Val Pro          35 40 45 Val Ser Lys Val Val Ser Thr Asp Glu Tyr Val Ala Arg Thr Asn Ile      50 55 60 Tyr Tyr His Ala Gly Thr Ser Arg Leu Leu Ala Val Gly His Pro Tyr  65 70 75 80 Phe Pro Ile Lys Lys Pro Asn Asn Asn Lys Ile Leu Val Pro Lys Val                  85 90 95 Ser Gly Leu Gln Tyr Arg Val Phe Arg Ile His Leu Pro Asp Pro Asn             100 105 110 Lys Phe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr Gln Arg         115 120 125 Leu Val Trp Ala Cys Val Gly Val Glu Val Gly Arg Gly Gln Pro Leu     130 135 140 Gly Val Gly Ile Ser Gly His Pro Leu Leu Asn Lys Leu Asp Asp Thr 145 150 155 160 Glu Asn Ala Ser Ala Tyr Ala Ala Asn Ala Gly Val Asp Asn Arg Glu                 165 170 175 Cys Ile Ser Met Asp Tyr Lys Gln Thr Gln Leu Cys Leu Ile Gly Cys             180 185 190 Lys Pro Pro Ile Gly Glu His Trp Gly Lys Gly Ser Pro Cys Thr Asn         195 200 205 Val Ala Val Asn Pro Gly Asp Cys Pro Pro Leu Glu Leu Ile Asn Thr     210 215 220 Val Ile Gln Asp Gly Asp Met Val Asp Thr Gly Phe Gly Ala Met Asp 225 230 235 240 Phe Thr Thr Leu Gln Ala Asn Lys Ser Glu Val Pro Leu Asp Ile Cys                 245 250 255 Thr Ser Ile Cys Lys Tyr Pro Asp Tyr Ile Lys Met Val Ser Glu Pro             260 265 270 Tyr Gly Asp Ser Leu Phe Phe Tyr Leu Arg Arg Glu Gln Met Phe Val         275 280 285 Arg His Leu Phe Asn Arg Ala Gly Ala Val Gly Glu Asn Val Pro Asp     290 295 300 Asp Leu Tyr Ile Lys Gly Ser Gly Ser Thr Ala Asn Leu Ala Ser Ser 305 310 315 320 Asn Tyr Phe Pro Thr Pro Ser Gly Ser Met Val Thr Ser Asp Ala Gln                 325 330 335 Ile Phe Asn Lys Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn Asn             340 345 350 Gly Ile Cys Trp Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr Thr         355 360 365 Arg Ser Thr Asn Met Ser Leu Cys Ala Ala Ile Ser Thr Ser Glu Thr     370 375 380 Thr Tyr Lys Asn Thr Asn Phe Lys Glu Tyr Leu Arg His Gly Glu Glu 385 390 395 400 Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Lys Ile Thr Leu Thr Ala                 405 410 415 Asp Val Met Thr Tyr Ile His Ser Met Asn Ser Thr Ile Leu Glu Asp             420 425 430 Trp Asn Phe Gly Leu Gln Pro Pro Pro Gly Gly Thr Leu Glu Asp Thr         435 440 445 Tyr Arg Phe Val Thr Ser Gln Ala Ile Ala Cys Gln Lys His Thr Pro     450 455 460 Pro Ala Pro Lys Glu Asp Pro Leu Lys Lys Tyr Thr Phe Trp Glu Val 465 470 475 480 Asn Leu Lys Glu Lys Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly                 485 490 495 Arg Lys Phe Leu Leu Gln Ala Gly Leu Lys Ala Lys Pro Lys Phe Thr             500 505 510 Leu Gly Lys Arg Lys Ala Thr Pro Thr Thr Ser Ser Thr Ser Thr Thr         515 520 525 Ala Lys Arg Lys Lys Arg Lys Leu Lys Leu Lys Pro Pro Thr Pro Pro     530 535 540 Pro Glu Pro Glu Thr 545 <210> 4 <211> 550 <212> PRT <213> Artificial Sequence <220> 223 fusion polypeptide HPV 18 L1 <400> 4 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro   1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg              20 25 30 Gly Ser Glu Phe Val Tyr Leu Pro Pro Pro Val Val Ala Arg Val Val          35 40 45 Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser Ile Phe Tyr His Ala Gly      50 55 60 Ser Ser Arg Leu Leu Thr Val Gly Asn Pro Tyr Phe Arg Val Pro Ala  65 70 75 80 Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys Val Ser Ala Tyr Gln Tyr                  85 90 95 Arg Val Phe Arg Val Gln Leu Pro Asp Pro Asn Lys Phe Gly Leu Pro             100 105 110 Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln Arg Leu Val Trp Ala Cys         115 120 125 Ala Gly Val Glu Ile Gly Arg Gly Gln Pro Leu Gly Val Gly Leu Ser     130 135 140 Gly His Pro Phe Tyr Asn Lys Leu Asp Asp Thr Glu Ser Ser His Ala 145 150 155 160 Ala Thr Ser Asn Val Ser Glu Asp Val Arg Asp Asn Val Ser Val Asp                 165 170 175 Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly Cys Ala Pro Ala Ile Gly             180 185 190 Glu His Trp Ala Lys Gly Thr Ala Cys Lys Ser Arg Pro Leu Ser Gln         195 200 205 Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn Thr Val Leu Glu Asp Gly     210 215 220 Asp Met Val Asp Thr Gly Tyr Gly Ala Met Asp Phe Ser Thr Leu Gln 225 230 235 240 Asp Thr Lys Cys Glu Val Pro Leu Asp Ile Cys Gln Ser Ile Cys Lys                 245 250 255 Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp Pro Tyr Gly Asp Ser Met             260 265 270 Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe Ala Arg His Phe Trp Asn         275 280 285 Arg Ala Gly Thr Met Gly Asp Thr Val Pro Gln Ser Leu Tyr Ile Lys     290 295 300 Gly Thr Gly Met Arg Ala Ser Pro Gly Ser Cys Val Tyr Ser Pro Ser 305 310 315 320 Pro Ser Gly Ser Ile Val Thr Ser Asp Ser Gln Leu Phe Asn Lys Pro                 325 330 335 Tyr Trp Leu His Lys Ala Gln Gly His Asn Asn Gly Val Cys Trp His             340 345 350 Asn Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser Thr Asn Leu         355 360 365 Thr Ile Cys Ala Ser Thr Gln Ser Pro Val Pro Gly Gln Tyr Asp Ala     370 375 380 Thr Lys Phe Lys Gln Tyr Ser Arg His Val Glu Glu Tyr Asp Leu Gln 385 390 395 400 Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu Thr Ala Asp Val Met Ser                 405 410 415 Tyr Ile His Ser Met Asn Ser Ser Ile Leu Glu Asp Trp Asn Phe Gly             420 425 430 Val Pro Pro Pro Thr Thr Thr Ser Leu Val Asp Thr Tyr Arg Phe Val         435 440 445 Gln Ser Val Ala Ile Thr Cys Gln Lys Asp Ala Ala Pro Ala Glu Asn     450 455 460 Lys Asp Pro Tyr Asp Lys Leu Lys Phe Trp Asn Val Asp Leu Lys Glu 465 470 475 480 Lys Phe Ser Leu Asp Leu Asp Gln Tyr Pro Leu Gly Arg Lys Phe Leu                 485 490 495 Val Gln Ala Gly Leu Arg Arg Lys Pro Thr Ile Gly Pro Arg Lys Arg             500 505 510 Ser Ala Pro Ser Ala Thr Thr Ser Ser Lys Pro Ala Lys Arg Val Arg         515 520 525 Val Arg Ala Arg Lys Val Asp Lys Leu Ala Asn Leu Pro His Leu Pro     530 535 540 Leu Asn Leu Lys His Xaa 545 550 <210> 5 <211> 1658 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding fusion polypeptide HPV 6 L1 <400> 5 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcat gtggcggcct 120 agcgacagca cagtatatgt gcctcctcct aaccctgtat ccaaagttgt tgccacggat 180 gcttatgtta ctcgcaccaa catattttat catgccagca gttctagact tcttgcagtg 240 ggacatcctt atttttccat aaaacgggct aacaaaactg ttgtgccaaa ggtgtcagga 300 tatcaataca gggtatttaa ggtggtgtta ccagatccta acaaatttgc attgcctgac 360 tcgtctcttt tcgatcccac aacacaacgt ttagtatggg catgcacagg cctagaggtg 420 ggcaggggac agccattagg tgtgggtgta agtggacatc ctttcctaaa taaatatgat 480 gatgttgaaa attcagggag tggtggtaac cctggacagg ataacagggt taatgtaggt 540 atggattata aacaaacaca attatgcatg gttggatgtg cccccccttt gggcgagcat 600 tggggtaaag gtaaacagtg tactaataca cctgtacagg ctggtgactg cccgccctta 660 gaacttatta ccagtgttat acaggatggc gatatggttg acacaggctt tggtgctatg 720 aattttgctg atttgcagac caataaatca gatgttccta ttgacatatg tggcactaca 780 tgtaaatatc cagattattt acaaatggct gcagacccat atggtgatag attatttttt 840 tttctacgga aggaacaaat gtttgccaga cattttttta acagggctgg cgaggtgggg 900 gaacctgtgc ctgatacact tataattaag ggtagtggaa atcgcacgtc tgtagggagt 960 agtatatatg ttaacacccc gagcggctct ttggtgtcct ctgaggcaca attgtttaat 1020 aagccatatt ggctacaaaa agcccaggga cataacaatg gtatttgttg gggtaatcaa 1080 ctgtttgtta ctgtggtaga taccacacgc agtaccaaca tgacattatg tgcatccgta 1140 actacatctt ccacatacac caattctgat tataaagagt acatgcgtca tgtggaagag 1200 tatgatttac aatttatttt tcaattatgt agcattacat tgtctgctga agtaatggcc 1260 tatattcaca caatgaatcc ctctgttttg gaagactgga actttgggtt atcgcctccc 1320 ccaaatggta cattagaaga tacctatagg tatgtgcagt cacaggccat tacctgtcaa 1380 aagcccactc ctgaaaagga aaagccagat ccctataaga accttagttt ttgggaggtt 1440 aatttaaaag aaaagttttc tagtgaattg gatcagtatc ctttgggacg caagtttttg 1500 ttacaaagtg gatatagggg acggtcctct attcgtacag gtgttaagcg ccctgctgtt 1560 tccaaagcct ctgctgcccc taaacgtaag cgcgccaaaa ctaaaagggt cgacaagctt 1620 gcaaacctcc cacacctccc cctgaacctg aaacataa 1658 <210> 6 <211> 1661 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding fusion polypeptide HPV 11 L1 <400> 6 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcat gtggcggcct 120 agcgacagca cagtatatgt gcctcctccc aaccctgtat ccaaggttgt tgccacggat 180 gcgtatgtta aacgcaccaa catattttat catgccagca gttctagact ccttgctgtg 240 ggacatccat attactctat caaaaaagtt aacaaaacag ttgtaccaaa ggtgtctgga 300 tatcaatata gagtgtttaa ggtagtgttg ccagatccta acaagtttgc attacctgat 360 tcatccctgt ttgaccccac tacacagcgt ttagtatggg cgtgcacagg gttggaggta 420 ggcaggggtc aacctttagg cgttggtgtt agtgggcatc cattgctaaa caaatatgat 480 gatgtagaaa atagtggtgg gtatggtggt aatcctggtc aggataatag ggttaatgta 540 ggtatggatt ataaacaaac ccagctatgt atggtgggct gtgctccacc gttaggtgaa 600 cattggggta agggtacaca atgttcaaat acctctgtac aaaatggtga ctgccccccg 660 ttggaactta ttaccagtgt tatacaggat ggggacatgg ttgatacagg ctttggtgct 720 atgaattttg cagacttaca aaccaataaa tcggatgttc cccttgatat ttgtggaact 780 gtctgcaaat atcctgatta tttgcaaatg gctgcagacc cttatggtga taggttgttt 840 ttttatttgc gaaaggaaca aatgtttgct agacactttt ttaatagggc cggtactgtg 900 ggggaacctg tgcctgatga cctgttggta aaagggggta ataacagatc atctgtagct 960 agtagtattt atgtacatac acctagtggc tcattggtgt cttcagaggc tcaattattt 1020 aataaaccat attggcttca aaaggctcag ggacataaca atggtatttg ctggggaaac 1080 cacttgtttg ttactgtggt agataccaca cgcagtacaa atatgacact atgtgcatct 1140 gtgtctaaat ctgctacata cactaattca gattataagg aatacatgcg ccatgtggag 1200 gagtttgatt tacagtttat ttttcaattg tgtagcatta cattatctgc agaagtcatg 1260 gcctatatac acacaatgaa tccttctgtt ttggaggact ggaactttgg tttatcgcct 1320 ccaccaaatg gtacactgga ggatacttat agatatgtac agtcacaggc cattacctgt 1380 cagaaaccca cacctgaaaa agaaaaacag gatccctata aggatatgag tttttgggag 1440 gttaacttaa aagaaaagtt ttcaagtgaa ttagatcagt ttccccttgg acgtaagttt 1500 ttattgcaaa gtggatatcg aggacggacg tctgctcgta caggtataaa gcgcccagct 1560 gtgtctaagc cctctacagc ccccaaacga aaacgtacca aaaccaaaaa ggtcgacaag 1620 cttgcaaacc tcccacacct ccccctgaac ctgaaacata a 1661 <210> 7 <211> 1650 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding fusion polypeptide HPV 16 L1 <400> 7 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcga gctccgtcga 120 caagtctact tgcctcctgt cccagtatct aaggttgtaa gcacggatga atatgttgca 180 cgcacaaaca tatattatca tgcaggaaca tccagactac ttgcagttgg acatccctat 240 tttcctatta aaaaacctaa caataacaaa atattagttc ctaaagtatc aggattacaa 300 tacagggtat ttagaataca tttacctgac cccaataagt ttggttttcc tgacacctca 360 ttttataatc cagatacaca gcggctggtt tgggcctgtg taggtgttga ggtaggtcgt 420 ggtcagccat taggtgtggg cattagtggc catcctttat taaataaatt ggatgacaca 480 gaaaatgcta gtgcttatgc agcaaatgca ggtgtggata atagagaatg tatatctatg 540 gattacaaac aaacacaatt gtgtttaatt ggttgcaaac cacctatagg ggaacactgg 600 ggcaaaggat ccccatgtac caatgttgca gtaaatccag gtgattgtcc accattagag 660 ttaataaaca cagttattca ggatggtgat atggttgata ctggctttgg tgctatggac 720 tttactacat tacaggctaa caaaagtgaa gttccactgg atatttgtac atctatttgc 780 aaatatccag attatattaa aatggtgtca gaaccatatg gcgacagctt atttttttat 840 ttacgaaggg aacaaatgtt tgttagacat ttatttaata gggctggtgc tgttggtgaa 900 aatgtaccag acgatttata cattaaaggc tctgggtcta ctgcaaattt agccagttca 960 aattattttc ctacacctag tggttctatg gttacctctg atgcccaaat attcaataaa 1020 ccttattggt tacaacgagc acagggccac aataatggca tttgttgggg taaccaacta 1080 tttgttactg ttgttgatac tacacgcagt acaaatatgt cattatgtgc tgccatatct 1140 acttcagaaa ctacatataa aaatactaac tttaaggagt acctacgaca tggggaggaa 1200 tatgatttac agtttatttt tcaactgtgc aaaataacct taactgcaga cgttatgaca 1260 tacatacatt ctatgaattc cactattttg gaggactgga attttggtct acaacctccc 1320 ccaggaggca cactagaaga tacttatagg tttgtaacat cccaggcaat tgcttgtcaa 1380 aaacatacac ctccagcacc taaagaagat ccccttaaaa aatacacttt ttgggaagta 1440 aatttaaagg aaaagttttc tgcagaccta gatcagtttc ctttaggacg caaattttta 1500 ctacaagcag gattgaaggc caaaccaaaa tttacattag gaaaacgaaa agctacaccc 1560 accacctcat ctacctctac aactgctaaa cgcaaaaaac gtaagctgaa gcttaaacct 1620 cccacacctc cccctgaacc tgaaacataa 1650 <210> 8 <211> 1649 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding fusion polypeptide HPV 18 L1 <400> 8 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcgt atatcttcca 120 cctccttctg tggcaagagt tgtaaatacc gatgattatg tgactcgcac aagcatattt 180 tatcatgctg gcagctctag attattaact gttggtaatc catattttag ggttcctgca 240 ggtggtggca ataagcagga tattcctaag gtttctgcat accaatatag agtatttagg 300 gtgcagttac ctgacccaaa taaatttggt ttacctgata ctagtattta taatcctgaa 360 acacaacgtt tagtgtgggc ctgtgctgga gtggaaattg gccgtggtca gcctttaggt 420 gttggcctta gtgggcatcc attttataat aaattagatg acactgaaag ttcccatgcc 480 gccacgtcta atgtttctga ggacgttagg gacaatgtgt ctgtagatta taagcagaca 540 cagttatgta ttttgggctg tgcccctgct attggggaac actgggctaa aggcactgct 600 tgtaaatcgc gtcctttatc acagggcgat tgcccccctt tagaacttaa aaacacagtt 660 ttggaagatg gtgatatggt agatactgga tatggtgcca tggactttag tacattgcaa 720 gatactaaat gtgaggtacc attggatatt tgtcagtcta tttgtaaata tcctgattat 780 ttacaaatgt ctgcagatcc ttatggggat tccatgtttt tttgcttacg gcgtgagcag 840 ctttttgcta ggcatttttg gaatagagca ggtactatgg gtgacactgt gcctcaatcc 900 ttatatatta aaggcacagg tatgcgtgct tcacctggca gctgtgtgta ttctccctct 960 ccaagtggct ctattgttac ctctgactcc cagttgttta ataaaccata ttggttacat 1020 aaggcacagg gtcataacaa tggtgtttgc tggcataatc aattatttgt tactgtggta 1080 gataccactc gcagtaccaa tttaacaata tgtgcttcta cacagtctcc tgtacctggg 1140 caatatgatg ctaccaaatt taagcagtat agcagacatg ttgaggaata tgatttgcag 1200 tttatttttc agttgtgtac tattacttta actgcagatg ttatgtccta tattcatagt 1260 atgaatagca gtattttaga ggattggaac tttggtgttc cccccccgcc aactactagt 1320 ttggtggata catatcgttt tgtacaatct gttgctatta cctgtcaaaa ggatgctgca 1380 ccggctgaaa ataaggatcc ctatgataag ttaaagtttt ggaatgtgga tttaaaggaa 1440 aagttttctt tagacttaga tcaatatccc cttggacgta aatttttggt tcaggctgga 1500 ttgcgtcgca agcccaccat aggccctcgc aaacgttctg ctccatctgc cactacgtct 1560 tctaaacctg ccaagcgtgt gcgtgtacgt gccaggaagg tcgacaagct tgcaaacctc 1620 ccacacctcc ccctgaacct gaaacataa 1649 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer HPV6L1F <400> 9 gaattcatgt ggcggcctag cgacagc 27 <210> 10 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer HPV6L1R <400> 10 gcatgagtcg acccttttag ttttggcgcg c 31 <210> 11 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer HPV11L1F <400> 11 gaattcatgt ggcggcctag cgacagc 27 <210> 12 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer HPV11L1R <400> 12 gcatgagtcg acctttttgg ttttggtacg ttt 33 <210> 13 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer HPV16L1F <400> 13 gtcgaccgat gcaggtgact tttatttaca tc 32 <210> 14 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer HPV16L1R <400> 14 aagcttcagc ttacgttttt tgcgtttagc 30 <210> 15 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer HPV18L1F <400> 15 gaattcatgt gcctgtatac acgggtcctg 30 <210> 16 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer HPV18L1R <400> 16 gcatgagtcg accttcctgg cacgtacacg cac 33

Claims (4)

N 말단에 히스티딘 택(Histidine tag)의 아미노산 서열,
C 말단에 시미안 바이러스 40 대형 T 항원(simian virus 40 large T-antigen)의 아미노산 서열 또는 이의 활성단편, 및
HPV 6 L1 항원, HPV11 L1 항원, HPV16 L1 항원 및 HPV18 L1 항원으로 구성된 군으로부터 선택된 HPV L1 항원의 아미노산 서열 또는 이의 활성단편을 포함하는 융합 폴리펩타이드가 결합된 비드 어레이용 비드;
표지자 표지된 2차 항체; 및
형광물질이 부착된 상기 표지자의 특이적 결합제를 포함하는 비드 어레이용 HPV 항체 검출용 키트.
Amino acid sequence of histidine tag at the N-terminus,
The amino acid sequence of the simian virus 40 large T-antigen at its C terminus or an active fragment thereof, and
Beads for bead arrays to which a fusion polypeptide comprising an amino acid sequence of an HPV L1 antigen selected from the group consisting of HPV 6 L1 antigen, HPV11 L1 antigen, HPV16 L1 antigen and HPV18 L1 antigen or an active fragment thereof is bound;
Marker labeled secondary antibodies; And
HPV antibody detection kit for the bead array comprising a specific binding agent of the marker attached to the fluorescent material.
제1항에 있어서, 상기 융합 폴리펩타이드의 아미노산 서열은 서열번호 1 내지 4로 구성된 군으로부터 선택된 아미노산 서열인, 키트.
The kit of claim 1, wherein the amino acid sequence of the fusion polypeptide is an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4.
제1항에 있어서, 상기 표지자는 비오틴이고 상기 표지자의 특이적 결합제는 스트렙타비딘인, 키트.
The kit of claim 1, wherein the marker is biotin and the specific binding agent of the marker is streptavidin.
제1항에 있어서, 상기 형광물질은 플루오레신, 이소티오시아네이트, 로다민, 피코에리트린, 피코시아닌, 알로피코시아닌, o-프탈데히드 또는 플루오레스카민인, 키트.
The kit of claim 1, wherein the fluorescent material is fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde or fluorescamine.
KR1020120053336A 2012-05-18 2012-05-18 Screening Kit for Human Papillomavirus antibody using fusion polypeptide HPV antigen KR101347288B1 (en)

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CN108872596A (en) * 2018-07-05 2018-11-23 重庆巴而思生物科技有限公司 A kind of ELISA detection kit of HPV16 L1 antibody
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