KR101347288B1 - Screening Kit for Human Papillomavirus antibody using fusion polypeptide HPV antigen - Google Patents

Abstract

The present invention relates to an HPV antibody screening method 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. A fusion polypeptide comprising an amino acid sequence of an HPV L1 antigen selected from the group consisting of a sequence or an active fragment thereof and an HPV 6 L1 antigen, an HPV11 L1 antigen, an HPV16 L1 antigen, and an HPV18 L1 antigen, or an active fragment thereof, said fusion polypeptide HPV antigen It relates to a method for detecting HPV antibody in a sample using the HPV antibody detection kit for bead array containing the fusion polypeptide HPV antigen. 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.

Description

Screening Kit for Human Papillomavirus antibody using fusion polypeptide HPV antigen}

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The polynucleotide encoding the fusion polypeptide may be a nucleotide sequence selected from the group consisting of SEQ ID NOs: 5-8.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Example 1 : HPV  6 L1 , HPV  11 L1 , HPV  16 L1  And HPV  18 L1  Construction of Expression Vectors for Gene Expression

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.

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.

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.

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 ℃.

SEQ ID NO: 9 Primer (HPV6L1F):

5'- GAATTC ATGTGGCGGCCTAGCGACAGC-3 '(includes EcoR I cut)

SEQ ID NO: 10 Primer (HPV6L1R):

5'- GCATGA GTCGAC CCTTTTAGTTTTGGCGCGC-3 '(Including Sal I cut)

SEQ ID NO: 11 Primer (HPV11L1F):

5'- GAATTC ATGTGGCGGCCTAGCGACAGC-3 'with EcoR I cut

SEQ ID NO: 12 Primer (HPV11L1R):

5'- GCATGA GTCGAC CTTTTTGGTTTTGGTACGTTT-3 'with Sal I cut

SEQ ID NO: 13 Primer (HPV16L1F):

5'- GTCGAC CGATGCAGGTGACTTTTATTTACATC-3 'with Sal I cut

SEQ ID NO: 14 Primer (HPV16L1R):

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

SEQ ID NO: 15 Primer (HPV18L1F):

5'- GAATTC ATGTGCCTGTATACACGGGTCCTG-3 '(Including EcoR I cleavage)

SEQ ID NO: 16 Primer (HPV18L1R):

5'- GCATGA GTCGAC CTTCCTGGCACGTACACGCAC-3 '(includes Sal I cut)

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).

Example  2: HPV  6 L1 , HPV  11 L1 , HPV  16 L1 , HPV  18 L1  Include fusion Of the polypeptide  Expression and Purification

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.

Fusion proteins expressed from the recombinant strains were purified as follows.

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.

Example  3: purified fusion The polypeptide  Containing Bead  For arrays Bead  making

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.

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.

Example  4: Multiplex Luminex  analysis

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.

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).

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).

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Claims (4)

Amino acid sequence of histidine tag at the N-terminus,
The amino acid sequence of the simian virus 40 large T-antigen at the C terminus, 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 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.
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.
The kit of claim 1, wherein the marker is biotin and the specific binding agent of the marker is streptavidin.
The kit of claim 1, wherein the fluorescent material is fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde or fluorescamine.

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