HK1124343B - Truncated human papillomavirus type 6 l1 proteins - Google Patents

Description

Truncated human papillomavirus type 6L1 protein

Technical Field

The invention relates to a truncated human papilloma virus 6 type L1 protein, a viroid particle composed of the protein, a vaccine containing the viroid particle and application of the vaccine in preventing condyloma acuminatum or HPV (especially HPV6) infection.

Background

Human papillomavirus HPV (human Papilomoavirus) belongs to the Papovaviridae (Papovaviridae) genus of Papillomavirus, which is a non-enveloped DNA virus. The viral genome is double-stranded closed-loop DNA, about 7.2-8 kb in size, and has 8 open frames. The genome can be divided into three regions according to the function: early region (E), about 4.5kb, encodes E1, E2, E4-E76 nonstructural proteins involved in viral replication, transcription and transformation; late region (L), about 2.5kb, encodes major capsid protein L1 and minor capsid protein L2; and the long regulation and control region (LCR) is positioned between the tail end of the L region and the initial end of the E region, has the length of about 800-900 bp, does not encode any protein, and contains DNA replication and expression regulation and control elements. The diameter of the virus particle is 45-55 nm, the nucleocapsid is 20-face-symmetric, and the virus particle comprises 72 shell particles which are composed of L1 and L2.

There are more than 90 subtypes of HPV, which are known at present and mainly cause wart lesions of skin and mucosa in human population. They can be further divided into 3 groups according to their relationship with tumorigenesis: (ii) a low or no oncogenic risk group comprising HPV6, 11, 39, 41, 42, 43; ② the intermediate carcinogenic risk group, including HPV31, 33, 35, 51, 52; ③ high cancer risk group, comprising HPV16, 18, 45, 56.

Epidemiological investigations have shown that infection of the anogenital mucosa with HPV such as HPV6, 11 is second only to chlamydia and trichomoniasis and third, a common sexually transmitted disease. And the lesions caused by HPV6, 11 account for about 90% of the total lesions. In the United states, the peak of HPV infection of the female genital tract is between 15 and 25 years of age and is closely related to the sexual behavior of infected individuals. In China, the peak period of the HPV infection rate of women is between 20 and 29 years old, and the infection rate is 1606.1/10 ten thousand. Women over the age of 35 have a progressively lower rate of HPV infection. However, since HPV infection is mostly subclinical infection, the infection rate is difficult to estimate accurately, but the estimated lifetime of the U.S. CDC accumulates approximately 10% of the risk of HPV infection. In addition, there is little information about male infections due to the difficulty in collecting large samples of male specimens and the less severe consequences of HPV infection in men. It is estimated, however, that the infection rate in men should be close to that in women. Whereas in the united states, visible condyloma acuminata is found in 1% of sexually active adult males. Therefore, the development of a safe and effective HPV6, 11 vaccine is an effective means for preventing sexually transmitted diseases.

The HPV L1 protein is a main capsid protein, has the molecular weight of 55-60 kDa and is a main target protein of an HPV vaccine. HPV L1 protein expressed in various expression systems forms viroid-like particles (VLPs) with morphological structures similar to native Virus particles without the aid of L2 protein. The virus-like particle has an icosahedral three-dimensional symmetrical structure and consists of 72 pentamers of L1 protein. It retains the natural epitope of virus particle, has strong immunogenicity, and can induce neutralizing antibody against homotypic HPV virus. (Kirnbauer, R., F. Booy, et al.1992Proc Natl Acad Sci U S A89 (24): 12180-4.) and, viroid does not carry viral nucleic acids, is not a potential carcinogenic hazard, and has good safety. Therefore, VLP vaccines have become a major direction for HPV vaccine development.

The key to HPV VLP vaccine development is the ability to efficiently prepare VLP samples in large quantities. The expression systems commonly used at present can be classified into eukaryotic expression systems and prokaryotic expression systems.

Examples of eukaryotic expression systems that are commonly used include poxvirus expression systems, insect baculovirus expression systems, and yeast expression systems. The natural conformation of the HPV L1 protein expressed in the eukaryotic expression system is little damaged, the VLP can be spontaneously formed, and the purified VLP can be obtained by only simple density gradient centrifugation, thereby providing great convenience for purification work. However, the expression level of the eukaryotic expression system is low, the culture cost is high, and great difficulty is brought to large-scale industrial production. The HPV vaccine Gardasil which is currently marketedThe saccharomyces cerevisiae expression system is adopted, the expression quantity is low, the production cost is high, and therefore the product price is higher, and the wide application of the product is influenced.

The expression of HPVL1 protein in prokaryotic expression system by using E.coli expression system has been reported. For example, the expression of HPV16L1 protein (Banks, L., G.Matlashewski, et al. (1987). J Gen Virol 68(Pt 12): 3081-9) using E.coli has been reported. However, since most of the HPV L1 protein expressed by E.coli loses its native conformation, protective antibodies against HPV cannot be generated. Or the protein can also obtain HPV VLP (Kelsall, S.R.and J.K.Kulski (1995). J Virol Methods 53 (1): 75-90) through steps of inclusion body purification, renaturation and the like, but the protein loss amount is large in the renaturation process, the yield is low, and the application on large-scale production is difficult. Although the HPV L1 protein can be expressed in Escherichia coli in a soluble manner in a correct conformation and dissolved in the lysate supernatant of the cells, the expression level is low, and the amount of the foreign protein in the supernatant is large, so that it is difficult to purify the target protein therefrom. Although there are reports in the literature that the expression level of L1 protein in the supernatant can be increased by GST fusion expression and the purification of target protein is facilitated (Li, m., t.p.cripte, et al (1997). J Virol 71 (4): 2988-95), the cleavage of fusion protein often requires expensive enzyme and cannot be applied to large-scale production.

Therefore, there is still a need in the art for a low cost induction of HPV l1 protein and viroid particles composed thereof against HPV protective antibodies, thereby enabling the large-scale industrial production of condyloma acuminatum vaccines.

Disclosure of Invention

The invention aims to provide a novel HPV6L1 protein, viroid particles composed of the same and a vaccine containing the viroid particles.

The inventor surprisingly found that a truncated HPV6L1 protein capable of inducing neutralizing antibodies against HPV6 can be obtained in an E.coli expression system, and the truncated HPV6L1 protein is purified to obtain the HPVL1 protein with high yield and at least 50% purity. The purified HPVL1 protein is further processed to obtain viroid particles capable of inducing protective antibodies against HPV6, and the invention is completed based on the invention.

Accordingly, in a first aspect the present invention relates to an HPV6L1 protein N-terminally truncated by 2, 3, 4 or 5 amino acids (compared to the wild-type HPV6L1 protein). Preferably the truncated protein has sequence 1, 2, 3, or 4, preferably sequence 1.

In yet another aspect, the invention relates to polynucleotides encoding the truncated proteins of the invention and vectors comprising the polynucleotides.

In a further aspect the invention relates to a cell comprising the vector described above.

The invention also relates to compositions comprising the truncated proteins or polynucleotides or vectors or cells described above.

Yet another aspect of the invention relates to an HPV 6-like virion, wherein the virion comprises an HPV6L1 protein N-terminally truncated by 2, 3, 4, or 5 amino acids, such as an HPV6L1 protein having the sequence 1, 2, 3, or 4, or consists of or is formed from an HPV6L1 protein N-terminally truncated by 2, 3, 4, or 5 amino acids, such as an HPV6L1 protein having the sequence 1, 2, 3, or 4.

In yet another aspect, the present invention relates to a method for obtaining HPV6L1 protein, which comprises expressing a truncated HPV6L1 gene fragment in an E.coli expression system, and purifying the lysed supernatant containing the truncated protein.

In a preferred embodiment, the method for obtaining HPV6L1 protein comprises

a) Expressing the truncated HPV6L1 gene segment in an Escherichia coli expression system,

b) the Escherichia coli expressing the truncated HPV6L1 protein is crushed in salt concentration of 100mM-600mM, separated to obtain supernatant,

c) reducing the salt concentration in the supernatant of b) to 100mM or less, to a minimum of 0, with water or a low salt solution, collecting the precipitate,

d) redissolving the precipitate from c) in a salt solution of 150mM to 2500mM, while adding a reducing agent, and isolating a solution containing truncated HPV6L1 protein having a purity of at least 50%.

More generally, the invention also relates to a method for obtaining an HPV1 protein, such as the HPV6L1 protein of the invention, comprising

a) Expressing HPVL1 gene coding HPVL1 protein in an E.coli expression system,

b) the Escherichia coli expressing HPVL1 protein was disrupted at a salt concentration of 100mM-600mM, separated to obtain a supernatant,

c) reducing the salt concentration in the supernatant of b) to 100mM or less, to a minimum of 0, with water or a low salt solution, collecting the precipitate,

d) redissolving the precipitate from c) in a 150mM-2500mM salt solution while adding a reducing agent, and isolating a solution comprising HPVL1 protein at least 50% pure.

The invention also relates to a vaccine for preventing condyloma acuminatum or HPV infection, which comprises the HPV6L1 protein virus particles. Preferably, the vaccine further comprises at least one viroid selected from the group consisting of HPV18L1 protein viroid, HPV11L1 protein viroid, HPV16L1 protein viroid, HPV31L1 protein viroid, HPV33L1 protein viroid, HPV45L1 protein viroid, HPV52L1 protein viroid and HPV58L1 protein viroid. The vaccine will typically also contain an excipient or carrier for the vaccine.

Preferably, the vaccine comprises: HPV 6-type virus particles and HPV 11-type virus particles, in particular, comprising a nucleotide sequence having the sequence of SEQ ID NO: 4 or HPV 6-like virus particles formed from the protein, and a polypeptide comprising an amino acid sequence having the amino acid sequence shown in SEQ ID NO: 7 or HPV 11-like virus particles formed from the protein. More preferably the vaccine further comprises HPV 16-like virions and HPV 18-like virions, in particular a vaccine comprising a polypeptide having the sequence of seq id NO: 8 or HPV 16-like virus particles formed from the protein, and a polypeptide comprising an amino acid sequence having the sequence shown in SEQ ID NO: 9 or HPV 18-like virus particles formed from the protein.

In a particularly preferred embodiment, the vaccine comprises: comprises a polypeptide having the sequence of SEQ ID NO: 4 or HPV 6-like virus particles formed from the protein, comprising a polypeptide having the amino acid sequence shown in SEQ ID NO: 7 or HPV 11-like virus particles formed from the protein, comprising a polypeptide having the amino acid sequence shown in SEQ ID NO: 8 or HPV 16-like virus particles formed from the protein, and a polypeptide comprising an amino acid sequence having the sequence shown in SEQ id no: 9 or HPV 18-like virus particles formed from the protein.

The invention further relates to application of the HPV6L1 protein or the viroid thereof in preparing a vaccine for preventing condyloma acuminatum or HPV infection.

The invention also relates to a method for preventing condyloma acuminatum or HPV infection, which comprises the step of administering a vaccine containing a preventive effective amount of HPV6L1 protein to a human or an animal needing to prevent condyloma acuminatum or HPV infection.

The invention also relates to a method for obtaining HPV6L1 protein viroid, which comprises the following steps:

e) the truncated HPV6L1 protein with the purity of at least 50 percent is further purified by chromatographic chromatography,

f) removing a reducing agent from the HPV6L1 protein obtained in the step e).

The invention also relates to a method for preparing a vaccine for preventing condyloma acuminatum or HPV infection, which comprises mixing the viroid particles with one or more optional HPV type viroid particles selected from HPV11, 16, 18, 31, 33, 45, 52 and 58 and a carrier or excipient for the vaccine.

Description and explanation of related terms in the present invention

According to the present invention, the term "E.coli expression system" means a system consisting of E.coli (strain) derived from commercially available sources, exemplified herein but not limited thereto: GI698, ER2566, BL21(DE3), B834(DE3), BLR (DE 3).

According to the present invention, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein expressed. The vector may be transformed, transduced or transfected into a host cell to obtain expression of the genetic material element carried by the vector in the host cell. By way of example, the carrier includes: a plasmid; bacteriophage; cosmids, and the like.

According to the present invention, the term "truncated HPV6L1 protein gene fragment" refers to the deletion of nucleotides encoding one or more amino acids at the 5 'end or 3' end of wild-type HPV6L1 protein gene (cDNA), wherein the full-length sequence of wild-type HPV6L1 protein gene is exemplified by, but not limited to, the following sequences in NCBI database: : AF067042.1, AF092932.1, L41216.1, X00203.1, and the like.

The term "truncated HPV6L1 protein" refers to a protein obtained by removing one or more amino acids from the N-and/or C-terminus of wild-type HPV6L1 protein, wherein examples of wild-type HPV6L1 protein include, but are not limited to, full-length L1 proteins encoded by AF067042.1, AF092932.1, L41216.1, X00203.1, etc. in NCBI database.

According to the present invention, the term "vaccine excipient or carrier" refers to a substance selected from one or more of, including but not limited to: pH regulator, surfactant, adjuvant, and ionic strength enhancer. For example, pH adjusting agents such as, but not limited to, phosphate buffers, surfactants include cationic, anionic or nonionic surfactants. By way of example but not limitation: tween-80. Adjuvants are exemplified by, but not limited to, aluminum hydroxide, freund's complete adjuvant. Ionic strength enhancers are exemplified by, but not limited to, sodium chloride.

According to the present invention, the term "chromatography" includes, but is not limited to: ion exchange chromatography (e.g., cation exchange chromatography), hydrophobic interaction chromatography, adsorption chromatography (e.g., hydroxyapatite chromatography), gel filtration (gel exclusion) chromatography, affinity chromatography.

According to the invention, the truncated HPV6L1 protein of the invention is preferably obtained as follows: subjecting E.coli expressing a truncated HPV6L1 protein to disruption in a buffer having a salt concentration of 100-600mM, preferably 200-500mM, separating the disrupted solution to obtain a supernatant, reducing the salt concentration of the obtained supernatant to a salt concentration of 100mM-0M with water or a low-concentration salt (usually lower than the salt concentration for disruption), and separating the precipitate in the supernatant having a salt concentration of as low as 100 mM-0; the precipitate is redissolved in a solution containing a reducing agent and a salt concentration of 150-2000mM, preferably above 200mM, and isolated to give a solution of truncated HPV6L1 protein having a purity of at least 50%, preferably at least 70%, more preferably at least 80%.

According to the present invention, in the method of obtaining the truncated HPV6L1 protein according to the present invention, the buffer refers to a solution that can maintain a stable pH value within a certain range, including, but not limited to, Tris buffer, phosphate buffer, HEPES buffer, MOPS buffer, and the like.

According to the present invention, the prokaryotic host cell disruption includes, but is not limited to, one or more of homogenizer disruption, sonication, milling, high pressure extrusion, lysozyme treatment;

according to the invention, in the method for truncating HPV6L1 protein obtained by the invention, the salts used include but are not limited to neutral salts, in particular alkali metal salts, ammonium salts, hydrochlorides, sulfates, bicarbonates, phosphates or hydrogen phosphates, in particular NaCl, KCl, NH₄Cl、(NH4)₂SO₄One or more of them. NaCl is preferred. Reducing agents used include, but are not limited to, DTT, 2-mercaptoethanol. Amounts used include, but are not limited to, 10mM to 100 mM.

According to the invention, the truncated HPV6L1 protein viroid of the invention is obtained as follows: the truncated HPV VL1 protein solution with the purity of at least 50% is further separated by chromatography, for example, to obtain a purified truncated HPV6L1 protein solution. And removing the reducing agent in the purified truncated HPV6L1 protein solution to obtain the viroid particles of the truncated HPV6L 1. Means for removing the reducing agent include, but are not limited to, techniques known in the art, such as dialysis, ultrafiltration or chromatography, and the like.

According to the invention, the truncated HPVL1 protein of the invention preferably has the sequence 1.

According to the present invention, the vaccine of the present invention may take a patient-acceptable form, including but not limited to oral administration or injection, preferably injection.

According to the invention, the vaccine of the invention is preferably used in a unit dosage form, wherein the amount of the viral particles of the truncated HPV6L1 protein class in the unit dosage form is 5 μ g-80 μ g, preferably 20 μ g-40 μ g.

Advantageous effects

The expression systems adopted in the preparation of the HPV viroid can be divided into eukaryotic expression systems and prokaryotic expression systems.

The natural conformation of the expressed HPVL1 protein in eukaryotic expression systems is less disrupted, VLPs can be formed spontaneously, and VLPs with the correct conformation can be obtained by simple purification. However, the baculovirus expression system and the yeast expression system adopted by the current eukaryotic expression system have the defects of low expression level, high culture cost and the like, and bring great difficulty to large-scale industrial production.

In a prokaryotic expression system, an escherichia coli expression system has the advantages of low culture cost and large expression quantity. However, the HPVL1 protein expressed in the E.coli expression system often lost the correct native conformation and was expressed in the pellet as inclusion bodies. Renaturation of proteins expressed in inclusion bodies is still a worldwide problem at present. The difficulty of renaturation, the inefficiency, makes it difficult to obtain VLPs of the correct conformation from inclusion bodies in large scale production, limited to small scale laboratory studies. Although HPVL1 can also be expressed in soluble form in the E.coli lysate supernatant in the correct conformation, the expression level is low, and it is difficult to purify HPVL1 from a wide variety of soluble proteins in the E.coli lysate supernatant, and purification by fusion expression, affinity chromatography, or other means is often necessary, which requires expensive enzymes and is difficult to industrially produce.

The invention expresses the N-end truncated HPV6L1 protein in an escherichia coli expression system, selectively precipitates and expresses the HPV6L1 protein in escherichia coli cracking supernatant by adopting a mild means, and further re-dissolves the HPV6L1 protein by adopting a saline buffer solution, so that the purity of the re-dissolved target protein is obviously improved on the premise of keeping the correct conformation of the HPV6L1 protein, and the re-dissolved target protein can be directly subjected to ion exchange chromatography and hydrophobic exchange chromatography purification to obtain pure protein. The purified truncated HPV6L1 truncated protein obtained by the steps can be assembled into viroid particles, has good immunogenicity, can induce high-titer neutralizing antibodies aiming at HPV6, prevents HPV6 from infecting human bodies, and is a good vaccine form. In addition, the truncated HPV6L1 protein adopted in the invention is easy to express in an Escherichia coli expression system while the antigenicity and the particle assembly capacity of the full-length HPV6L1 protein are kept, and the adopted purification method does not need to use expensive enzyme and has low cost. And the target protein is not subjected to a severe denaturation and renaturation process in the conformation in the purification process, has small loss and can be applied to large-scale industrial production.

These and other aspects of the invention will be apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are incorporated by reference in their entirety.

Drawings

FIG. 1 shows the result of SDS polyacrylamide gel electrophoresis of HPV6N3C-L1 protein in steps a) -d) of the method of the invention. 1, cracking the supernatant; 2, HPV6N3C-L1 subjected to tangential flow precipitation; 3, HPV6N3C-L1 resuspended in resuspension. The result shows that the purity of the HPV6N3C-L1 protein reaches about 70 percent after the steps of precipitation and redissolution.

FIG. 2 shows the result of SDS polyacrylamide gel electrophoresis of HPV6N3C-L1 obtained in step d) after further purification in step e) of the present invention. 1, carrying out the step e) purification of the invention to obtain HPV6N3C-L1, and loading 10 mu L of the HPV6N 3C-L1; 2, HPV6N3C-L1 purified by step e) of the invention was loaded in 20. mu.l. The result shows that the purity of the HPV6N3C-L1 protein purified by the step e) reaches about 98 percent.

FIG. 3 shows the result of transmission electron microscopy (50,000 fold) of HPV6N 3C-L1-like virions obtained in step f). A large number of viroid particles with the radius of about 25nm can be seen in the visual field, and the particle size is consistent with the theoretical size and is uniform.

FIG. 4 shows dynamic light scattering observations of HPV6N 3C-L1-like virus particles obtained in step f). The result shows that the hydration molecular kinetic radius of the HPV6N3C-L1 virus particle is 24.70nm, and the particle assembly percentage is 100%.

FIG. 5 shows serum neutralizing antibody titers at various stages after HPV6N 3C-L1-like virions were inoculated into sheep. In the figure, the arrows indicate the immunization times. After one week of priming, the titer of the neutralizing antibody is obviously increased, and after one-time boosting, the titer of the neutralizing antibody can be reachedTo 10⁷-10⁸To a higher level.

FIG. 6 shows serum neutralizing antibody titers at various stages after inoculation of rabbits with HPV6N 3C-L1-like virions. In the figure, the arrows indicate the immunization times. After one week of priming, the titer of the neutralizing antibody is obviously increased, and after one-time boosting, the titer of the neutralizing antibody can reach 10⁶To a higher level.

FIG. 7 shows the variation of the HPV6 and HPV11 neutralizing antibody titers in the sera of the mice at different times after the bivalent HPV6/11 vaccination in example 5. The immunization program was 0, 2W (weeks). After the first immunization, the titer of the HPV6 and HPV11 neutralizing antibodies is obviously increased, and after the first boosting immunization, the titer of the antibodies can reach 1 × 10⁴-1×10⁵。

FIG. 8 shows the variation of the neutralizing antibody titers against HPV6, HPV11, HPV16, HPV18 in the serum at different times after the tetravalent vaccination of HPV6/11/16/18 in example 5 in mice. The immunization program was 0, 2W (weeks). After the first immunization, the titer of neutralizing antibodies of HPV6, HPV11, HPV16 and HPV18 is obviously increased, and after the first boosting immunization, the titer of the antibodies can reach 1 × 10⁵-1×10⁶。

FIG. 9 shows the HPV6L1 proteins HPV6N2C-L1, HPV6N4C-L1, HPV6N5C-L1 truncated at the N-terminus by 2, 4, or 5 amino acids, respectively, in steps a) -e) of the method of the invention (the amino acid sequences are shown in SEQ ID NO: 2. 3, 4) in the SDS-polyacrylamide gel electrophoresis. 1, molecular weight Marker; 2, carrying out the purification of the steps a) to e) of the invention to obtain HPV6N2C-L1, and loading 10 μ L of the HPV6N 2C-L1; 3, carrying out the purification of the steps a) to e) of the invention to obtain HPV6N4C-L1, and loading 10 mu L of the HPV6N 4C-L1; 4, carrying out the purification of the steps a) to e) of the invention to obtain 10 mu L of HPV6N 5C-L1; the result shows that the protein purity of the HPV6L1 protein with 2, 4 or 5 amino acids truncated at the N end, HPV6N2C-L1, HPV6N4C-L1 and HPV6N5C-L1 are all up to about 98 percent after the treatment of the steps a) to e).

FIG. 10 shows the result of transmission electron microscopy (50,000 fold) of HPV6L1 protein HPV6N2C-L1, HPV6N4C-L1, HPV6N5C-L1 type virions, which are obtained by steps a) -f) and are truncated by 2, 4, or 5 amino acids at the N-terminus, respectively. 1, transmitting electron microscope observation (50,000 times) results of HPV6N2C-L1 viroid particles obtained by the steps a) to f); 2, transmitting electron microscope observation (50,000 times) results of HPV6N4C-L1 viroid particles obtained by the steps a) to f); 3, the result of transmission electron microscope observation (50,000 times) of HPV6N5C-L1 viroid particles obtained by the steps a) to f); the results show that a large number of viroid particles with a radius of about 25nm are visible in the visual field, and the particle size is consistent with the theoretical size and is uniform.

FIG. 11 shows the dynamic light scattering observations of HPV6L1 proteins HPV6N2C-L1, HPV6N4C-L1, HPV6N5C-L1 viroid particles with 2, 4, or 5 amino acids truncated at the N-terminus obtained through steps a) -f), respectively. 1, carrying out dynamic light scattering observation on HPV6N2C-L1 viroid particles obtained by the steps a) -f); 2, carrying out dynamic light scattering observation on the HPV6N4C-L1 viroid particles obtained in the steps a) -f); 3, observing the dynamic light scattering observation result of the HPV6N5C-L1 viroid particles obtained by the steps a) to f); the result shows that the hydration molecular dynamics radius of the HPV6N2C-L1, HPV6N4C-L1 and HPV6N5C-L1 viroid particles is about 25nm, and the particle assembly percentage is 100%.

Sequence of

The invention is further illustrated by the following examples. These examples are not limiting.

Example 1: expression of truncated HPV6L1 protein having sequence 1

Preparation of HPV6L1 Gene fragment used as template

The HPV-6L1 gene was synthesized in its full length by Shanghai Boya. The total length of the synthesized gene fragment is 1503bp, and the sequence thereof is sequence 5. On the basis of the artificially synthesized HPV-6L1 gene full-length fragment, the template of the truncated HPV6L1 protein is prepared.

Construction of non-fusion expression vector of truncated HPV6L1 Gene

The HPV6L1 full-length gene fragment synthesized in the previous step was used as a template for the second PCR reaction. With 6N 3F: 5' -CAT ATgCCT AGC GAC AGC ACA GTA TA-3 '(SEQ ID NO: 10) as a forward primer, wherein a restriction enzyme NdeI site is introduced into the 5' end of the forward primer, the sequence of the NdeI site is CAT ATG, and the ATG is an initiation codon in an Escherichia coli system; 6 CR: 5' -GTC GACTTA CCT TTT AGT TTT GGC GC-3 '(SEQ ID NO: 11) is a reverse primer, the 5' end of which incorporates a SalI site for a restriction enzyme. The PCR reaction was carried out in a PCR thermal cycler (Biometra T3) under the following conditions:

the specific DNA fragment of about 1.5kb is obtained by amplification. The PCR product was ligated with a commercially available pMD18-T vector (manufactured by TAKARA) and subjected to NdeI/SalI restriction to obtain pMD18-T-HPV6N3C-L1, which is a positive clone into which the truncated HPV6L1 gene was inserted.

The M13(+)/(-) primer is utilized by Shanghai Boya bioengineering company to measure that the target nucleotide sequence inserted into the pMD18-T-HPV6N3C-L1 plasmid is sequence 6, and the coded amino acid sequence is sequence 1: the protein corresponding to the sequence is HPV6L1 protein with the N end truncated by 3 amino acids and the C end not truncated, and is named as HPV6N 3C-L1.

The pMD18-T-HPV6N3C-L1 plasmid is subjected to NdeI/SalI digestion to obtain a truncated HPV6N3C-L1 gene fragment. This fragment was then ligated with an NdeI/SalI-cut pTrxFus prokaryotic expression vector (from Invitrogen) without the other fusion polypeptide immediately after the initial amino acid Met, since the fusion protein had been cleaved off. NdeI/SalI enzyme digestion identification is carried out to obtain a positive expression clone pTRX-HPV-6N3C-L1 inserted into the gene fragment of the L1 protein. mu.L of pTRX-HPV-6N3C-L1 plasmid (0.15mg/ml) was transferred to 40. mu.L of competent E.coli GI698 prepared by calcium chloride method (purchased from Invitrogen) and plated on ampicillin (final concentration 100mg/ml, the same applies hereinafter) resistant solid CAA medium (6g Na)₂HPO₄，3g KH₂PO₄，0.5g NaCl，1g NH₄Cl, 20g casein hydrolysate, 0.095g MgCl₂1.5g of agar powder is dissolved in 900ml of deionized water, 20ml of 50% glycerol is added, and the mixture is kept still and cultured for 10 to 12 hours at the temperature of 30 ℃ until single colonies are clear and distinguished. Single colonies were picked up in tubes containing 4mL of liquid IMC medium for ampicillin resistance, cultured at 25 ℃ for 10 hours with shaking at 220 rpm, and 1mL of the resulting suspension was lyophilized at-70 ℃.

Large scale expression of HPV6N3C-L1

Taking out the Escherichia coli freeze-dried strain with the recombinant plasmid pTRX-HPV6N3C-L1 from-70 ℃, inoculating 50ml of IMC liquid culture medium with ampicillin resistance, culturing at 200rpm and 30 ℃ for about 8 hours, then transferring into 10 bottles of 500ml culture medium, inoculating 5ml of bacterial liquid into each bottle, culturing at 200rpm and 30 ℃ in a shaking bottle overnight.

Main instrument, Shanghai Baoxing biological company 50L fermenter

Correcting the pH electrode of the fermentation tank, preparing 30 liters of culture medium, filling the culture medium into the fermentation tank, sterilizing the culture medium at the temperature of 121 ℃ for 30 minutes, and correcting the dissolved oxygen electrode by taking the initial stirring speed of 100rpm after aeration during fermentation as the zero point before aeration.

Preparing for feeding, wherein the concentration of casein hydrolysate is 30% (30g to 100ml), the glucose is 50% (50g to 100ml), and the mixture is sterilized at 121 ℃ for 20 minutes.

The next day, 10 bottles of seed solution (5L) were placed in a fermentor at 30 ℃ and pH7.0, and the stirring rate and aeration rate were manually adjusted to maintain dissolved oxygen at above 40%.

Feeding materials, mixing 50% glucose and 30% casein hydrolysate according to the mass ratio of 2: 1 of solute.

The flow acceleration rate is as follows:

100% is 25ml/min

The first hour: 5 percent;

and (3) in the second hour: 10 percent;

and (3) for the third hour: 20 percent;

and (4) in the fourth hour: 40 percent;

60% after the fifth hour

When the culture is carried out until the bacterial concentration reaches about 10 OD600, the culture temperature is reduced to 25 ℃ and 4g of tryptophan is added for induction culture for 4 hours. The final concentration was about 40 (OD600) and the cells were centrifuged to collect about 2.5kg

The formulation of the IMC medium was as follows (1 liter):

Na2HPO4	6g
		KH2PO4	3g
NaCl	0.5g
		NH4Cl	1g
casein hydrolysate	20g
		MgCl2	0.095g

Example 2: obtaining of HPV6N3C-L1 with a purity of about 70%

The cells were resuspended in a proportion of 1g of cells to 10mL of lysate (20mM Tris buffer pH7.2, 300mM NaCl) and disrupted 5 times at 600bar pressure using An APV homogenizer (An Invensys Group product). JA-14 was spun at 13500rpm (30000g), centrifuged for 15min, and the supernatant was collected and examined by 10% SDS-polyacrylamide gel electrophoresis, at which point the purity of HPV6N3C-L1 in the supernatant was about 10%. The supernatant was dialyzed using an CENTRASETTE 5 tangential flow device (PALL product) using a membrane with a molecular weight cut-off of 30kDa, a 10mM phosphate buffer pH 6.0 buffer as the dialysis solution, a dialysis volume of three times the volume of the supernatant, a pressure of 0.5psi during operation, a flow rate of 500mL/min and a tangential flow rate of 200 mL/min. After sufficient dialysis, the pellet was harvested by centrifugation at 9500rpm (12000g) in JA-10 rotor (Beckman J25 high speed centrifuge) for 20min, resuspended in 1/10 supernatant volumes of 10mM phosphate buffer pH7.0, 10mM DTT, 300mM NaCl and stirred for 30 min. JA-14 rotor (Beckman J25 high speed centrifuge), 13500rpm (30000g), centrifuge for 20min, centrifuge to obtain supernatant, filter the sample with 0.22 μm pore size filter, and purify the sample by cation exchange chromatography. A150. mu.L filtered sample was added to a 6X Loading Buffer of 30. mu.L. Mixing, water bathing at 80 deg.C for 10min, and electrophoresing 10 μ L in 10% SDS-polyacrylamide gel at 120V for 120 min. The electrophoretic bands were then visualized by Coomassie blue staining and the results are shown in FIG. 1. SDS-PAGE analysis shows that after the HPV6N3C-L1 protein is precipitated and redissolved, the target protein is purified and enriched, and the purity reaches about 70 percent.

Example 3: chromatographic purification of HPV6N3C-L1

Cation exchange chromatography purification of HPV6N3C-L1

An instrument system: preparative liquid chromatography system model AKTA explorer 100, manufactured by GE Healthcare Promega Amersham Pharmacia.

Chromatography medium: SP Sepharose 4 Fast Flow.

Column volume: 5.5cm × 20 cm.

Buffer solution: 20mM phosphate buffer pH7.0, 10mM DTT

20mM phosphate buffer pH7.010mM DTT 2M NaCl.

Flow rate: 25mL/min

Detector wavelength: 280nm

The sample is 3L HPV6N3C-L1 solution with purity of about 70%

The elution procedure was: 200mM NaCl elutes the hybrid protein, 500mM NaCl elutes the target protein, and 500mM NaCl elutes the product, and the total yield of HPV6N3C-L1 purified sample 900 mL.

CHT-II (hydroxyapatite chromatography) purification of HPV6N3C-L1

An instrument system: preparative liquid chromatography system model AKTA explorer 100, manufactured by GE Healthcare Promega Amersham Pharmacia.

Chromatography medium: CHT-II (purchased from Bio-RAD)

Column volume: 5.5cm × 20cm

Buffer solution: 10mM phosphate buffer pH7.0, 10mM DTT, 0.5M NaCl.

Flow rate: 20 mL/min.

Detector wavelength: 280 nm.

The samples were: SP Sepharose 4 Fast Flow 500mM NaCl eluate

The elution procedure was: direct recovery of penetrations containing the protein of interest

The breakthrough product was collected to obtain 1000mL of purified HPV6N3C-L1 sample. 150 μ L of HPV6N3C-L1 purified by the method of the present example was sampled, mixed with 30 μ L of 6X Loading Buffer, and subjected to electrophoresis at 120V for 120min in 10 μ L of 10% SDS-polyacrylamide gel after being placed in a 80 ℃ water bath for 10 min. The electrophoretic bands were then visualized by Coomassie blue staining and the results are shown in FIG. 2. As can be seen from the results of electrophoresis, the concentration of the target protein was about 0.7mg/ml, and the SDS-PAGE staining purity was more than 98%.

Example 4: assembly of HPV6N3C-L1 type virus particles

The instrument system is CENTRASETTE 5 tangential flow system produced by PALL; the membrane-bound molecular weight is 30 kDa; the sample is HPV6N 3C-L11000 ml obtained in example 3.

Concentration of the sample: the tangential flow rate of the system was adjusted to 50mL/min and the sample was concentrated to a total volume of 800 mL.

Renaturation of the sample: renaturation buffer (20mM PB pH 6.0, 2mM CaCl2, 2mM MgCl) 10L₂0.5M NaCl, 0.003% Tween-80) was well exchanged for sample buffer. The pressure was 0.5psi during the operation of the tangential flow device, the tangential flow rate was 10mL/min, and after the renaturation buffer had been exchanged, the exchange was carried out with storage buffer (20L PBS: 20mM PB pH 6.5, 0.5M NaCl) in a volume of 20L. The pressure was 0.5psi at run time and the tangential flow rate was 25 mL/min. For all liquidAfter the body exchange is finished, a sample is aseptically filtered by using a PALL 0.20 mu m filter to obtain HPV6N3C-L1 virus particles, and the particles are stored at 4 ℃ for later use.

Example 5: morphological detection and immunogenicity determination of HPV6N3C-L1 VLP

Transmission electron microscope observation of HPV6N3C-L1 virus particles

The instrument is a 100kV transmission electron microscope produced by Japan Electron company, and the magnification is 100,000 times. HPV6N3C-L1 virus particles are negatively stained with 2% phosphotungstic acid (pH7.0), and fixed on a charcoal-sprayed copper net for observation. As a result, as shown in FIG. 3, it can be seen that the sample obtained in example 4 contains a large number of viroid particles having a radius of about 25nm, and has a uniform size and a hollow morphology.

Dynamic light scattering observation of HPV6N3C-L1 virus-like particles

The instrument is a DynaPro MS/X type dynamic light scattering instrument (containing a temperature controller) manufactured by Protein Solutions in the United states, and the used algorithm is a Regulation algorithm. The sample was the sample obtained in example 4. The samples were filtered through a 0.22 μm filter and measured. The measurement results are shown in FIG. 4. The results showed that the hydration molecular kinetic radius of HPV6N3C-L1 VLP was 25.46 nm.

Establishment of HPV6 pseudovirus neutralization cell model

Since HPV is difficult to culture in vitro and has strong host specificity, it is difficult to propagate in a non-human host, and a suitable animal model is lacking. Therefore, in order to be able to rapidly assess the immunoprotection of HPV vaccines, it is necessary to establish an effective in vitro neutralization experimental model.

Pseudoviruses (pseudoviruses) in vitro infection model: the characteristic that HPV VLP can non-specifically package nucleic acid is utilized, and HPV pseudovirus is formed by expressing L1 and L2 proteins of HPV in cells, and wrapping episome virus DNA in cells or exogenously introduced reporter plasmid. Specific methods include recombinant virus expression system method and multi-plasmid co-transfection method (Yeager, M.D., Aste-Amezaga, M.et al (2000) Virology (278) 570-7).

The invention adopts a multi-plasmid cotransfection method, and adopts the following improvements aiming at an HPV system: establishes an optimized calcium phosphate transfection method for 293FT cells, can obtain the transfection efficiency of more than 90 percent, and is beneficial to large-scale production. The expression plasmid of the HPV structural protein optimized by the codon is obtained, can efficiently express HPV L1 and L2 genes in mammalian cells, and is beneficial to efficiently assembling pseudoviruses.

Construction of HPV pseudovirus

Plasmid p6L1h carrying HPV6L1 gene, plasmid p6L2h carrying HPV6L2 gene, and plasmid pN31-EGFP carrying green fluorescent protein gene were purified separately by CsCl density gradient centrifugation (these plasmids were given by professor NIH John T.Schiller). The CsCl density gradient centrifugation plasmid purification method is referred to molecular cloning: third edition. Briefly: the plasmid was transformed into E.coli DH 5. alpha. and a single colony was inoculated into 500mL of LB medium and shake-cultured at 37 ℃ for 16 hours. 9000g was centrifuged for 5min to collect the cells. To each 1000mL of the harvested cells, 40mL of solution I (50mM glucose, 25mM Tris-Cl (pH8.0), 10mM EDTA (pH 8.0)) and 2mL of RNase A (1. mu.g/. mu.L), 40mL of solution II (0.2M NaOH, 1% SDS), 48mL of solution III (60.0 mL of 5M potassium acetate, 11.5mL of glacial acetic acid, and 28.5mL of deionized water) were added in this order. Standing on ice for 10min, centrifuging at 4 deg.C for 20min at 15000g, mixing the supernatant with 0.6 times volume of isopropanol, centrifuging at 15000g for 30min, discarding the supernatant, washing the precipitate with 70% ethanol for 1 time, dissolving the precipitate with TE, and determining DNA content. CsCl (1.01 g CsCl per gram DNA) was dissolved in the DNA solution, 100uL of 10mg/mL ethidium bromide solution was added, and the mixture was centrifuged at 62000rpm using a Beckman NVT65 rotor at 20 ℃ for 10 hr. The closed loop DNA bands were collected with a syringe needle and the extraction was repeated 4 times with equal volumes of isoamyl alcohol. Adding 3 times volume of water and 8 times volume of absolute ethyl alcohol, 20000g, centrifuging at 4 deg.C for 30min, and collecting DNA precipitate. The DNA precipitate was dissolved in 1mL of TE after 1-pass washing with 75% ethanol. The concentration of the DNA solution was determined and stored in aliquots at-20 ℃.

The purified p6L1h, p6L2h, pN31-EGFP were co-transfected with 293FT cells (Invitrogen) grown in 10cm cell culture dishes using the calcium phosphate method. Calcium phosphate transfection method: 40ug each of p6L1h, p6L2h, pN31-EGFP was added to 1mL of HEPES solution (containing 1M HEPES 125uL at pH 7.3 per 50mL of deionized water, stored at 4 ℃) and 1mL of 0.5mol/LCaCl₂A mixed solution of the solutions was mixed, and 2mL of a 2 XHeBS solution (0.28M NaCl (16.36g), 0.05M HEPES (11.9g), 1.5mM Na) was added dropwise₂HPO₄(0.213g) was dissolved in 1000mL of deionized water and stored at-70 ℃ pH 6.96, and the mixture was left to stand at room temperature for 1min, and then the mixture was placed in a 10cm cell culture dish containing 293FT cells, after 6hr, the stock culture was discarded, and 10mL of complete culture medium (Invitrogen) was added. After transfection for 48hr, the medium was discarded, washed 2 times with PBS, cells were scraped off and collected, and the cells were counted every 10 th⁸Each cell was lysed with 1mL of lysate (0.25% Brij58, 9.5mM MgCl)₂) And (4) resuspending. After lysis, centrifugation is carried out for 10min at 5000g, the supernatant is collected, 5M NaCl (final concentration: 850mM) is added, and pseudovirus liquid is obtained, and the pseudovirus liquid is subpackaged into small parts and stored at the temperature of minus 20 ℃.

293FT cells (Invitrogen) were plated in 96-well cell culture plates (1.5X 10)⁴Hole/bore). After 5hr, a neutralization experiment was performed, and serum samples to be tested were serially diluted in duplicate with 10% DMEM, respectively, and 50 μ L of each serum sample was mixed with 50 μ L of the pseudovirus solution (moi ═ 0.1) prepared above diluted in 10% DMEM. After incubation for 1h at 4 ℃, the cells are respectively added into a 96-well cell culture plate pre-paved with 293FT cells, after culture for 72h at 37 ℃, the approximate neutralization titer of each sample is determined by fluorescence observation, then the infection rate of each well cell is detected by a flow cytometer (EPICS XL, Beckman Coulter company, USA), and the accurate neutralization titer of the monoclonal antibody or the polyclonal antiserum is calculated. The infection rate is the percentage of the number of cells in the positive zone of the cell sample minus the percentage of the number in the positive zone of the uninfected control cell sample.

The infection inhibition rate (1-infection rate of blocked wells/infection rate of unblocked wells) × 100%.

The antibody neutralization titer was defined as: maximum dilution times above 50% inhibition of infection were achieved. Monoclonal or polyclonal antibodies that achieve greater than 50% inhibition of infection after 50-fold dilution are considered to have neutralizing capacity.

Immunoprotective evaluation of animals immunized with HPV6 VLP vaccine

Rabbit: common grade, female, 6-8 weeks old, purchased from Guangxi province disease prevention and control center and raised in the center. The HPV6N3C-L1 virosome prepared in example 4 was prepared by mixing the same amount of Freund's complete adjuvant with primary immunization and the same amount of Freund's incomplete adjuvant with booster immunization by intramuscular injection at a primary immunization dose of 100 ug/dose, and then boosted once every 4 and 10 weeks, respectively, at a booster immunization dose of 50 ug/dose. After autoimmunity, peripheral venous blood is extracted weekly, serum is separated, and the serum is stored for detection.

Sheep: common grade, female, 6-8 weeks old, purchased from Guangxi province disease prevention and control center and raised in the center. The HPV6N3C-L1 viroid prepared in example 4 was prepared by mixing the same amount of Freund's complete adjuvant with primary immunization and the same amount of Freund's incomplete adjuvant with booster immunization by intramuscular injection at a primary immunization dose of 1 mg/dose, followed by booster injections at a booster dose of 0.5 mg/dose at 4, 10 and 18 weeks, respectively. After autoimmunity, peripheral venous blood is extracted weekly, serum is separated, and the serum is stored for detection.

The neutralizing titers of the above antisera were evaluated in the above pseudovirus neutralizing cell model experiment, as shown in FIGS. 5 and 6. The result shows that the HPV6N3C-L1 virus particles (except for Freund's adjuvant used in experiments, commercial or self-made aluminum hydroxide or aluminum phosphate adjuvant) obtained in the embodiment 4 of the invention are mixed to be prepared into vaccines, have good immunogenicity, can induce high-titer neutralizing antibodies in animals, and can be used as vaccines for preventing HPV infection.

Immunoprotection evaluation of mice immunized with HPV6/11 bivalent vaccine

The animals used for immunization were 4 SPF-grade BALB/c mice aged 4-5 weeks. HPV6N5C-L1 and HPV11N4C-L1 viroid granules were prepared in a similar manner as described in examples 1-4 of the present invention. The above 2 types of virus particles: HPV6N5C-L1 and HPV11N4C-L1 are mixed in a ratio of 1: 2 (weight ratio) so that the concentration of the two types of virus particles after mixing is 40, 80 mug/ml. Then, for priming, add equal volume of Freund's complete adjuvant and mix well. The booster immunization is prepared by mixing with an equal volume of Freund's incomplete adjuvant. The immunization mode is intramuscular injection, and the primary immunization dose is 10 mu g of HPV6N5C-L1 virus particles and 20 mu g of HPV11N4C-L1 virus particles. Thereafter, the mixture was boosted once every 2 weeks at a booster dose of 20. mu.g/HPV 6N 5C-L1-type virus particles and 40. mu.g/HPV 11N 4C-L1-type virus particles. After the self-immunization, peripheral venous blood was taken weekly, serum was separated, and neutralizing antibody titers against HPV6 and HPV11 pseudovirions in immunized mice were detected, respectively, as described in example 5. The detection results are shown in fig. 7, and the results show that the bivalent vaccine of HPV6 and HPV11 prepared by mixing two types of virus particles of HPV6N5C-L1 and HPV11N4C-L1 obtained by the method of examples 1 to 4 of the present invention has good immunogenicity, can induce high-titer neutralizing antibodies against HPV6 and HPV11 in animals, and can be used as an effective vaccine for preventing HPV6/HPV11 infection (except for the fowls adjuvant used in the examples, the vaccine can be prepared by mixing two types of virus particles of HPV16N5C-L1 and HPV11N4C-L1 prepared by the present invention with commercial or self-made aluminum hydroxide or aluminum phosphate adjuvant).

The corresponding amino acid sequence of the HPV11N4C-L1 virus particle is (SEQ ID NO: 7):

immunoprotection evaluation of mice immunized with HPV6/11/16/18 tetravalent vaccine

Immunizing animals: SPF-grade BALB/c mice 4-5 weeks old. Viroid particles of HPV6N5C-L1, HPV11N4C-L1, HPV16N30C-L1, and HPV18N65C-L1 were prepared in a similar manner to that described in examples 1-4 of the present invention. The above 4 types of virus particles: HPV6N5C-L1, HPV11N4C-L1, HPV16N30C-L1 and HPV18N65C-L1 are mixed according to the weight ratio of 1: 2: 1, so that the concentration of the four types of virus particles after mixing is 40, 80, 80 and 40 mu g/ml. Then, for priming, add equal volume of Freund's complete adjuvant and mix well. The booster immunization is prepared by mixing with an equal volume of Freund's incomplete adjuvant. The immunization mode is intramuscular injection, the primary immunization dose is 10 mu g/body of HPV6N5C-L1, 10 mu g/body of HPV18N65C-L1 virus particles, and 20 mu g/body of HPV11N4C-L1 and 20 mu g/body of HPV16N30C-L1 virus particles. Thereafter, the cells were boosted once every 2 weeks at a booster dose of 20. mu.g/cell for HPV6N5C-L1, HPV18N65C-L1 type virus particles, HPV11N4C-L1, and HPV16N30C-L1 type virus particles, respectively. After the self-immunization, peripheral venous blood was taken weekly, serum was separated, and neutralizing antibody titers against HPV6, HPV11, HPV16, HPV18 pseudovirions in immunized mice were detected, respectively, as described in example 5. The detection results are shown in FIG. 8, and the results show that the HPV6, HPV11, HPV16 and HPV18 tetravalent vaccines prepared by mixing HPV6N5C-L1, HPV11N4C-L1, HPV16N30C-L1 and HPV18N65C-L1 viroid particles obtained by the method of examples 1-4 of the present invention have good immunogenicity, can induce high-titer neutralizing antibodies against HPV6, HPV11, HPV16 and HPV18 in animals, and can be used as effective vaccines for preventing HPV6/HPV11/HPV16/HPV18 infection (besides Fowler's adjuvant used in the examples, the vaccines can be prepared by mixing HPV6N5C-L1, HPV11N4C-L1, HPV16N30C-L1 and HPV18N 65-L65C-L1 viroid particles with commercial or aluminum hydroxide or self-made adjuvant).

The L1 amino acid sequence of the HPV6N5C-L1 viroid is shown in SEQ ID NO: 4.

the L1 amino acid sequence of the HPV16N30C-L1 virus-like particle is (SEQ ID NO: 8):

the L1 amino acid sequence of the HPV18N65C-L1 virus-like particle is (SEQ ID NO: 9):

the amino acid sequence corresponding to the HPV11N4C-L1 virus particle is shown above (SEQ ID NO: 7).

Example 6:

according to the technology adopted in the embodiments 1-5 of the invention, the truncated HPV6L1 protein with the sequences 2, 3 and 4 is prepared, and the truncated protein can be purified to obtain the protein with the purity of more than 98 percent and assembled into viroid particles with the radius of about 25 nm. The results are shown in fig. 9, 10 and 11.

Sequence listing

Claims (19)

1. An HPV6L1 protein truncated at the N-terminus by 2, 3, 4, or 5 amino acids.
2. 2. The protein of claim 1, wherein the amino acid sequence is seq id No. 1, 2, 3, or 4.
3. 3. The protein of claim 1, wherein the amino acid sequence is seq id No. 1.
4. 4. A polynucleotide encoding a protein according to any one of claims 1 to 3.
5. 5. A vector comprising the polynucleotide of claim 4.
6. 6. A cell comprising the vector of claim 5.
7. 7. A composition comprising a protein according to any one of claims 1 to 3.
8. 8. An HPV 6-like virosome, wherein the virosome comprises a protein according to any one of claims 1-3 or is formed by a protein according to any one of claims 1-3.
9. 9. A method for preparing a protein according to any one of claims 1 to 3, comprising

   a) Expressing HPV L1 gene encoding HPV L1 in an E.coli expression system,

   b) the Escherichia coli expressing HPV L1 protein is crushed in salt concentration of 100mM-600mM, separated to obtain supernatant,

   c) reducing the salt concentration in the supernatant of b) to 100mM or less, to a minimum of 0, with water or a low salt solution, collecting the precipitate,

   d) redissolving the precipitate from c) in a 150mM-2500mM salt solution while adding a reducing agent, and isolating a solution containing HPV L1 protein at least 50% pure.
10. 10. A vaccine for the prevention of condyloma acuminata or HPV infection, comprising: (1) the HPV 6-type virion of claim 8, (2) optionally at least one HPV-type virion selected from the group consisting of types 11, 16, 18, 31, 33, 45, 52, and 58, and (3) a vaccine excipient or carrier.
11. 11. The vaccine of claim 10, wherein said at least one is 2, 3 or 4.
12. 12. The vaccine of claim 10, wherein said optional at least one HPV-type viral particle selected from the group consisting of types 11, 16, 18, 31, 33, 45, 52, and 58 is an HPV L1-type viral particle.
13. 13. The vaccine of claim 10, wherein the vaccine comprises HPV 6-type virus particles and HPV 11-type virus particles.
14. 14. The vaccine of claim 13, wherein said HPV 6-like virions and HPV 11-like virions are chimeric viruses comprising a nucleotide sequence whose amino acid sequence is SEQ ID NO: 4 and a HPV 6-like virus particle comprising a protein of SEQ ID NO: 7, and HPV 11-like virus particles.
15. 15. The vaccine of claim 13 or 14, wherein the vaccine further comprises HPV 16-type virus particles and HPV 18-type virus particles.
16. 16. The vaccine of claim 15, wherein said HPV 16-like virions and HPV 18-like virions are chimeric viruses comprising a nucleotide sequence whose amino acid sequence is SEQ ID NO: 8 and HPV 16-like virus particles comprising the protein of SEQ ID NO: 9, and HPV 18-like virus particles.
17. 17. Use of a protein according to any one of claims 1 to 3 or a viroid-like-particle according to claim 8 for the preparation of a vaccine for the prevention of condyloma acuminata or HPV infections.
18. 18. A method for obtaining HPV6L1 proteinaceous virosomes, comprising:

    e) further purifying the HPV6L1 protein of claim 1 or 2 or 3 in a purity of at least 50% by chromatography,

    f) removing a reducing agent from the HPV6L1 protein obtained in the step e).
19. 19. A method for preparing a vaccine for the prevention of condyloma acuminata or HPV infection, comprising mixing the viroid particles of claim 8 with optionally one or more HPV type viroid particles selected from HPV11, 16, 18, 31, 33, 45, 52, and 58, and a vaccine carrier or excipient.