Papillomavirus chimeric protein and application thereof

Abstract

Provided is a papillomavirus chimeric protein, the skeleton thereof being a papillomavirus L1 protein or a mutant thereof, at least one human papillomavirus 33-type L2 protein or mutant polypeptide thereof being embedded on the skeleton. The present papillomavirus chimeric protein can be used for preparing a vaccine for preventing papillomavirus infections and infection induced disease.

Claims

1. A chimeric papillomavirus protein, comprising: a papillomavirus HPV16 L1 protein or a mutant of the papillomavirus HPV16 L1 protein as a scaffold; and at least one polypeptide from a HPV33 L2 protein or from a mutant of the HFV33 L2 protein inserted into the scaffold, wherein the at least one polypeptide is shown as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6, the at least one polypeptide is inserted into a surface region of the HPV16 L1 protein or the mutant of the HPV16 L1 protein, and the at least one polypeptide is selected from the group consisting of: the polypeptide from the HPV33 L2 protein shown as SEQ ID NO: 1 or SEQ ID NO: 2 is inserted into the HPV16 L1 protein or the mutant of the HPV16 L1 protein between amino acid positions 136 and 137, after deletion of amino acid 135-138 region from the HPV16 L1 protein or the mutant of the HPV16 L1 protein, the polypeptide from the HPV33 L2 protein shown as SEQ ID NO: 5 or SEQ ID NO: 6 is inserted into the HPV16 L1 protein or the mutant of the HPV16 L1 protein between amino acid positions 134 and 139, and after deletion of amino acid 431-432 region from the HPV16 L1 protein or the mutant of the HPV16 L1 protein, the polypeptide from the HPV33 L2 protein shown as SEQ ID NO: 2 or SEQ ID NO: 3 is inserted into the HPV16 L1 protein or the mutant of the HPV16 L1 protein between amino acid positions 430 and 433.

2. The chimeric papillomavirus protein according to claim 1, wherein the HPV16 L1 protein or the mutant of the HPV16 L1 protein is shown as SEQ ID NO: 4 or the amino acid sequence of SEQ ID NO: 4 with 31 amino acid deletion at the C-terminus.

3. A polynucleotide encoding the chimeric papillomavirus protein according to claim 1.

4. A polymer composed of papillomavirus capsid protein, wherein the polymer is a chimeric pentamer or chimeric virus-like particle, which contains the chimeric papillomavirus protein of claim 1 or is consisted of the chimeric papillomavirus protein of claim 1.

5. A method for preventing HPV infection or HPV infection-associated diseases, comprising administering a prophylactically effective amount of the chimeric papillomavirus protein of one of claims 1 and 2 or the chimeric pentamer or chimeric virus-like particle of claim 4 to a subject.

6. A vaccine for preventing papillomavirus infection or infection-associated diseases, comprising the polymer of claim 4, adjuvants, and excipients or vehicles for the vaccine.

7. The polynucleotide of claim 3, wherein the sequence of the polynucleotide is optimized according to the codon usage preference of E. coli or insect cells.

8. The vaccine of claim 6, further comprising at least one virus-like particle or chimeric virus-like particle derived from mucosal and/or cutaneous HPV.

9. The vaccine of claim 6, wherein the adjuvant is a human adjuvant.

10. The vaccine of claim 9, wherein the human adjuvant is a composition selected from the group consisting of: a composition comprising Alum adjuvant, oil-in-water emulsion or water-in-oil emulsion and TLR agonist, a composition comprising aluminum hydroxide adjuvant or aluminum phosphate adjuvant, polyinosinic-polycytidic acid adjuvant and stabilizing agent, and a composition comprising MF59 adjuvant, polyinosinic-polycytidic acid adjuvant and stabilizing agent.

11. A vector comprising the polynucleotide of claim 3 or claim 7.

12. An isolated host cell comprising the vector of claim 11.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A-1B: Expression of chimeric proteins in E. coli and insect cells described in Example 5. The results showed that all the 12 chimeric proteins could be highly expressed in E. coli or insect cells.

(2) FIG. 1A: Expression of chimeric proteins in E. coli. Lane 1.HPV16L1DE.sub.136-137/33dE; Lane 2. HPV16L1DE.sub.136-137/33dEs; Lane 3. HPV16L1DE.sub.135-138/33dE; Lane 4. HPV16L1DE.sub.135-138/33dEs; Lane 5. HPV16L1h4/33dE; Lane 6. HPV16L1h4/33dEs.

(3) FIG. 1B: Expression of chimeric proteins in insect cells. Lane 1. HPV16L1CDE.sub.136-137/33dE; Lane 2. HPV16L1CDE.sub.136-137/33dEs; Lane 3. HPV16L1CDE.sub.135-138/33dE; Lane 4. HPV16L1CDE.sub.135-138/33dEs; Lane 5. HPV16L1Ch4/33dE; Lane 6. HPV16L1Ch4/33dEs.

(4) FIG. 2A-2B: DLS analysis of purified cVLPs described in Example 6. The results showed that the hydrodynamic diameters of HPV16L1CDE.sub.135-138/33dE and HPV 16L1CDE.sub.135-138/33dEs were 91.6 nm and 97.9 nm respectively. The chimeric proteins were 100% assembled into VLPs.

(5) FIG. 2A: HPV16L1CDE.sub.135-138/33dE; FIG. 2B: HPV16L1CDE.sub.135-138/33dEs

(6) FIG. 3A-3F: Transmission electron microscopy (TEM) analysis of purified cVLPs described in Example 7. A large number of VLPs were observed in the field of view. The particles were quite uniform in size. The cVLPs comprising L2 polypeptide in DE region were about 50 nm in diameter, which is similar to the diameter of VLP of L1 protein. The cVLPs comprising L2 polypeptide in h4 region were smaller in diameter, about 35-40 nm. Bar=200 nm.

(7) FIG. 3A: HPV16L1CDE.sub.136-137/33DE; FIG. 3B: HPV16L1CDE.sub.136-137/33dEs; FIG. 3C: HPV16L1CDE.sub.135-138/33DE; FIG. 3D: HPV16L1CDE.sub.135-138/33dEs; FIG. 3E: HPV16L1Ch4/33DE; FIG. 3F: HPV16L1Ch4/33dEs.

DETAILED DESCRIPTION OF THE INVENTION

(8) The present invention will now be further illustrated by the following non-limiting examples. It is well known to those skilled in the art that many modifications may be made to the present invention without departing from the spirit of the invention. Such modifications are also within the scope of the invention. The following examples are only illustrative of the present invention and should not be construed as limiting the scope of the invention, since the embodiments are necessarily varied. The terminology used in this specification is for the purpose of illustrating particular embodiments only, and is not to be taken by way of limitation. The scope of the invention is defined in the appended claims.

(9) Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. The preferred methods and materials of the present invention are described below, but any methods and materials similar or equivalent to those described in this invention may be used to practice or test the present invention. The following methods unless otherwise specified, are the conventional methods or methods described in the specification of products. Experimental materials used in the absence of special instructions, can be readily obtained from commercial companies. All publications mentioned in this specification are hereby incorporated by reference to disclose and describe the methods and/or materials in the publications.

EXAMPLE 1

Synthesis of the Genes of Chimeric L1 Proteins and the Construction of the Expression Vectors

(10) 12 chimeric L1 proteins are listed below:

(11) 1) Chimeric L1 protein HPV16 L1DE.sub.136-137/33dE: The scaffold of HPV16 L1DE.sub.136-137/33dE is full-length HPV16 L1 protein (sequence shown as SEQ ID No. 4), and aa. 16-37 polypeptide of HPV33 L2 protein was directly inserted into the DE loop of HPV16 L1 protein between aa. 136/137 (insertion sequence shown as SEQ ID No. 1). The polynucleotide encoding HPV16 L1DE.sub.136-137/33dE was optimized according to the codon usage preference of E. coli, and the coding sequence was constructed by inserting E. coli codon-optimized gene of HPV33 L2 protein aa. 16-37 (sequence shown as SEQ ID No. 9) between nucleotide (nt.) 408/409 of E. coli codon-optimized HPV16 L1 scaffold gene (sequence shown as SEQ ID No. 8).

(12) 2) Chimeric L1 protein HPV16 L1DE.sub.136-137/33dEs: The scaffold of HPV16 L1DE.sub.136-137/33dEs is full-length HPV16 L1 protein (sequence shown as SEQ ID No. 4), and aa. 17-32 polypeptide of HPV33 L2 protein was directly inserted into the DE loop of HPV16 L1 protein between aa. 136/137 (insertion sequence shown as SEQ ID No. 2). The polynucleotide encoding HPV16 L1DE.sub.136-137/33dEs was optimized according to the codon usage preference of E. coli, and the coding sequence was constructed by inserting E. coli codon-optimized gene of HPV33 L2 protein aa. 17-32 (sequence shown as SEQ ID No.10) between nt.408/409 of E. coli codon-optimized HPV16 L1 scaffold gene (sequence shown as SEQ ID No. 8).

(13) 3) Chimeric L1 protein HPV16 L1DE.sub.135-138/33dE: The scaffold of HPV16 L1DE.sub.135-138/33dE is full-length HPV16 L1 protein (sequence shown as SEQ ID No. 4), and aa. 135-139 region of HPV16 L1 protein was substituted by the aa. 16-37 polypeptide of HPV33 L2 protein, which contains linkers, i.e. the aa. 16-37 polypeptide with linkers was inserted into the HPV16 L1 protein between aa. 134/139 by non-equal length substitution, the amino acid sequence of the inserted fragment was shown as SEQ ID No. 5, wherein the N-terminal linker of HPV33 L2 aa. 16-37 polypeptide was G (glycine) P (proline) linker, and the C-terminal linker was P (proline) linker. The polynucleotide encoding HPV16 L1DE.sub.135-138/33dE was optimized according to the codon usage preference of E. coli, and the coding sequence was constructed by deleting nt.403-414 from E. coli codon-optimized HPV16 L1 gene (sequence shown as SEQ ID No. 8), and inserting sequence SEQ ID No.11 between nt.402/415 of the codon-optimized HPV16 L1 gene.

(14) 4) Chimeric L1 protein HPV16 L1DE.sub.135-138/33dEs: The scaffold of HPV16 L1DE.sub.135-138/33dEs is full-length HPV16 L1 protein (sequence shown as SEQ ID No. 4), and aa. 135-139 region of HPV16 L1 protein was substituted by the aa. 17-32 polypeptide of HPV33 L2 protein, which contains linkers, i.e. aa. 17-32 polypeptide with linkers was inserted into the HPV16 L1 protein between aa. 134/139 by non-equal-length substitution, the amino acid sequence of the inserted fragment was shown as SEQ ID No. 6, wherein the N-terminal linker of HPV33 L2 aa. 17-32 polypeptide was G (glycine) P (proline) linker, and the C-terminal linker was P (proline) linker. The polynucleotide encoding HPV16 L1DE.sub.135-138/33dEs was optimized according to the codon usage preference of E. coli, and the coding sequence was constructed by deleting nt.403-414 from E. coli codon-optimized HPV16 L1 gene (sequence shown as SEQ ID No. 8), and inserting sequence SEQ ID No.12 between nt.402/415 of the codon-optimized HPV16 L1 gene.

(15) 5) Chimeric L1 protein HPV16 L1h4/33dE: The scaffold of HPV16 L1h4/33dE is full-length HPV16 L1 protein (sequence shown as SEQ ID No. 4), and aa. 17-37 polypeptide of HPV33 L2 protein was inserted into the aa. 430-433 region of HPV16 L1 h4 region by non-equal length substitution. That is deleting the aa. 431-432 region from HPV16 L1 protein, and fusing the aa. 17-37 polypeptide with linkers between aa. 430/434 of HPV16 L1 protein. The amino acid sequence of the inserted fragment was shown as SEQ ID No. 3. The polynucleotide encoding HPV16 L1h4/33dE was optimized according to the codon usage preference of E. coli, and the coding sequence was constructed by deleting nt. 1291-1296 from E. coli codon-optimized HPV16 L1 gene (sequence shown as SEQ ID No. 8), and inserting sequence SEQ ID No.13 between nt. 1290/1297 of codon-optimized HPV16 L1 gene.

(16) 6) Chimeric L1 protein HPV16 L1h4/33dEs: The scaffold of HPV16 L1h4/33dE is full-length HPV16 L1 protein (sequence shown as SEQ ID No. 4), and aa. 17-32 polypeptide of HPV33 L2 protein was inserted into the aa. 430-433 region of HPV16 L1 h4 region by non-equal length substitution. That is deleting the aa. 431-432 region from HPV16 L1 protein, and fusing the aa. 17-32 polypeptide with linkers between aa. 430/434 of HPV16 L1. The amino acid sequence of inserted fragment was shown as SEQ ID No. 2. The polynucleotide coding for HPV16 L1h4/33dEs was optimized according to the codon usage preference of E. coli, and the coding sequence was constructed by deleting nt. 1291-1296 from E. coli codon-optimized HPV16 L1 gene (sequence shown as SEQ ID No. 8), and inserting sequence SEQ ID No.10 between nt. 1290/1297 of codon-optimized HPV16 L1 gene.

(17) 7) Chimeric L1 protein HPV16L1CDE.sub.136-137/33dE: The scaffold of HPV16L1CDE.sub.136-137/33dE is C-terminal truncated HPV16 L1 protein (deleting 31 amino acids from the sequence shown as SEQ ID No. 4 at the C-terminus), and aa. 16-37 polypeptide of HPV33 L2 protein was directly inserted into the DE loop of truncated HPV16 L1 protein between aa. 136/137 (insertion sequence shown as SEQ ID No. 1). The polynucleotide encoding HPV16 L1CDE.sub.136-137/33dE was optimized according to the codon usage preference of insect cells, and the coding sequence was constructed by inserting insect cell codon-optimized gene of HPV33 L2 protein aa. 16-37 (sequence shown as SEQ ID No.15) between nt.408/409 of insect cell codon-optimized HPV16 L1 scaffold gene (sequence shown as SEQ ID No. 14).

(18) 8) Chimeric L1 protein HPV16 L1CDE.sub.136-137/33dEs: The scaffold of HPV16 L1CDE.sub.136-137/33dEs is C-terminal truncated HPV16 L1 protein (deleting 31 amino acids from the sequence shown as SEQ ID No. 4 at the C-terminus), and aa. 17-32 polypeptide of HPV33 L2 protein was directly inserted into the DE loop of truncated HPV16 L1 protein between aa. 136/137 (insertion sequence shown as SEQ ID No. 2). The polynucleotide encoding HPV16L1CDE.sub.136-137/33dEs was optimized according to the codon usage preference of insect cells, and the coding sequence was constructed by inserting insect cell codon-optimized gene of HPV33 L2 protein aa. 17-32 (sequence shown as SEQ ID No.16) between nt.408/409 of insect cell codon-optimized HPV16 L1 scaffold gene (sequence shown as SEQ ID No. 14).

(19) 9) Chimeric L1 protein HPV16 L1CDE.sub.135-138/33dE: The scaffold of HPV16 L1CDE.sub.135-138/33dE is C-terminal truncated HPV16 L1 protein (deleting 31 amino acids from sequence shown as SEQ ID No. 4 at the C-terminus), and aa. 135-139 region of the truncated HPV16 L1 protein was substituted by the aa. 16-37 polypeptide of HPV33 L2 protein, which contains linkers, i.e. the aa. 16-37 polypeptide with linkers was inserted into the truncated HPV16 L1 protein between aa. 134/139 by non-equal length substitution, the amino acid sequence of inserted fragment was shown as SEQ ID No. 5, wherein the N-terminal linker of HPV33 L2 aa. 16-37 polypeptide was G (glycine) P (proline) linker, and the C-terminal linker was P (proline) linker. The polynucleotide encoding HPV16 L1CDE.sub.135-138/33dE was optimized according to the codon usage preference of insect cells, and the coding sequence was constructed by deleting nt.403-414 from the insect cell codon-optimized HPV16 L1 gene (sequence shown as SEQ ID No. 14), and inserting sequence SEQ ID No.17 between nt.402/415 of the codon-optimized HPV16 L1 gene.

(20) 10) Chimeric L1 protein HPV16 L1CDE.sub.135-138/33dEs: The scaffold of HPV16 L1CDE.sub.135-138/33dEs is C-terminal truncated HPV16 L1 protein (deleting 31 amino acids from the sequence shown as SEQ ID No. 4 at the C-terminus), and aa. 135-139 region of the truncated HPV16 L1 protein was substituted by aa. 17-32 polypeptide of HPV33 L2 protein, which contains linkers, i.e. aa. 17-32 polypeptide with linkers was inserted into the truncated HPV16 L1 between aa. 134/139 by non-equal length substitution, the amino acid sequence of inserted fragment was shown as SEQ ID No. 6, wherein the N-terminal linker of HPV33 L2 aa. 17-32 polypeptide was G (glycine) P (proline) linker, and the C-terminal linker was P (proline) linker. The polynucleotide encoding HPV16 L1CDE.sub.135-138/33dEs was optimized according to the codon usage preference of insect cells, and the coding sequence was constructed by deleting nt.403-414 from the insect cell codon-optimized HPV16 L1 gene (sequence shown as SEQ ID No. 14), and inserting sequence SEQ ID No.18 between nt.402/415 of the codon-optimized HPV16 L1 gene.

(21) 11) Chimeric L1 protein HPV16 L1Ch4/33dE: The scaffold of HPV16L1 Ch4/33dE is C-terminal truncated HPV16 L1 protein (deleting 31 amino acids from the sequence shown as SEQ ID No. 4 at the C-terminus), and aa. 17-37 polypeptide of HPV33 L2 protein was inserted into the aa. 430-433 region of truncated HPV16 L1 h4 region by non-equal length substitution. That is deleting the aa. 431-433 region from HPV16 L1 protein, and fusing the aa. 17-37 polypeptide of HPV33 L2 protein between aa. 430/434 of truncated HPV16 L1. The amino acid sequence of inserted fragment was shown as SEQ ID No. 3. The polynucleotide encoding HPV 16L1Ch4/33dE was optimized according to the codon usage preference of insect cells, and the coding sequence was constructed by deleting nt. 1291-1296 from insect cell codon-optimized HPV16 L1 gene (sequence shown as SEQ ID No. 14), and inserting sequence SEQ ID No. 19 between nt. 1290/1297 of the codon-optimized HPV16 L1 gene.

(22) 12) Chimeric L1 protein HPV16 L1Ch4/33dEs: The scaffold of HPV16 L1Ch4/33dEs is C-terminal truncated HPV16 L1 protein (deleting 31 amino acids from the sequence shown as SEQ ID No. 4 at the C-terminus of), and aa. 17-32 polypeptide of HPV33 L2 protein was inserted into the aa. 430-433 region of truncated HPV16 L1 h4 region by non-equal length substitution. That is deleting the aa. 431-432 region of HPV16 L1 protein, and fusing aa. 17-32 polypeptide of HPV33 L2 protein between aa. 430/434 of truncated HPV16 L1 protein. The amino acid sequence of inserted fragment was shown as SEQ ID No. 2. The polynucleotide coding for HPV16 L1Ch4/33dEs was optimized according to the codon usage preference of insect cells, and the coding sequence was constructed by deleting nt. 1291-1296 from insect cell codon-optimized HPV16 L1 gene (sequence shown as SEQ ID No. 14), and inserting sequence SEQ ID No. 16 between nt. 1290/1297 of the codon-optimized HPV16 L1 gene.

(23) Chimeric L1 genes were optimized according to the codon usage preference of E. coli or insect cells respectively, and optimized genes were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

(24) Each gene optimized according to codon usage preference of E. coli was digested by NdeI/XhoI enzymes, and were inserted into commercially available expression vector pET22b (Novagen). Each gene optimized according to codon usage preference of insect cells were digested by EcoRI/XbaI enzymes and were inserted into commercially available expression vector pFastBac1 (Invitrogen). The resulting recombinant chimeric L1 gene expression vectors were listed below: pET22b-16L1DE.sub.136-137/33dE; pET22b-16L1DE.sub.136-137/33dEs; pET22b-16L1DE.sub.135-138/33dE; pET22b-16L1DE.sub.135-138/33dEs; pET22b-16L1h4/33dE; pET22b-16L1h4/33dEs; pFastBac1-16L1CDE.sub.136-137/33dE; pFastBac 1-16L1CDE.sub.136-137/33dEs; pFastBac 1-16L1CDE.sub.135-138/33dE; pFastBac 1-16L1CDE.sub.135-138/33dEs; pFastBac1-16L1Ch4/33dE; pFastBac1-16L1Ch4/33dEs. Methods of digestion, ligation, and cloning used above are disclosed and are described, for example, in patent CN 101293918 B.

EXAMPLE 2

Construction of the Recombinant Bacmids and Recombinant Baculoviruses Containing the Chimeric L1 Protein Genes

(25) E. coli DH10BAC competent cells were transformed with recombinant expression vectors containing the chimeric L1 genes (pFastBac 1-16L1CDE.sub.136-137/33dE, pFastBac1-16L1CDE.sub.136-137/33dEs, pFastBac 1-16L1CDE.sub.135-138/33dE, pFastBac1-16L1CDE.sub.135-138/33dEs, pFastBac1-16L1Ch4/33dE, pFastBac1-16L1Ch4/33dEs), and recombinant bacmids were selected. The recombinant baculoviruses were then produced by transfecting Sf9 cells with the selected recombinant bacmids which propagating in the Sf9 cells. Methods of recombinant bacmid selection and recombinant baculovirus production are well-known and are described, for example, in patent CN101148661 B.

EXAMPLE 3

Expression of Chimeric L1 Protein Genes in Sf9 Cells

(26) Sf9 cells were infected with recombinant baculoviruses containing the above mentioned 6 chimeric L1 genes respectively, and the expression of the chimeric L1 proteins was carried out. The cells were cultured for 88 hours at 27 C., and were harvested by centrifuging at 3000 rpm for 15 min, and the supernatant was discarded. The cell pellets were washed with PBS and then used for expression analysis and purification. Methods of infection and expression are disclosed, for example, in patent CN 101148661 B.

EXAMPLE 4

Expression of Chimeric L1 Protein Genes in E. coli

(27) E. coli BL21(DE3) competent cells were transformed with recombinant expression vectors containing the chimeric L1 genes (pET22b-16L1DE.sub.136-137/33dE, pET22b-16L1DE.sub.136-137/33dEs, pET22b-16L1DE.sub.135-138/33dE, pET22b-16L1DE.sub.135-138/33dEs, pET22b-16L1h4/33dE, pET22b-16L1h4/33dEs).

(28) The transformed clones were inoculated into 3 ml of LB medium containing ampicillin and cultured overnight at 37 C. The overnight culture was added to LB medium at the ratio of 1:100, incubated at 37 C. for about 3 hours, until the OD600 reached 0.8-1.0. Then IPTG was added to medium at a final concentration of 0.5 M. The cells were cultured at 16 C. for 12 h, and then were harvested by centrifugation.

EXAMPLE 5

Expression Analysis of Chimeric L1 Protein

(29) Cells expressing different chimeric L1 proteins described in Example 3 and Example 4 were resuspended with PBS at a concentration of 110.sup.6 cells/200 l PBS and mixed with 50 l 6 Loading Buffer. Then the mixture was denatured at 75 C. for 8 min 10 l of denatured samples were loaded for SDS-PAGE and Western blotting analysis respectively. The results were shown in FIG. 1. All 12 recombinant chimeric L1 proteins were expressed effectively in E. coli or insect cells expression system. The molecular weight of HPV16L1DE.sub.136-137/33dE,HPV 16L1DE.sub.136-137/33dEs, HPV16L1DE.sub.135-138/33dE, HPV16L1DE.sub.135-138/33dEs, HPV16L1h4/33dE, HPV16L1h4/33dEs was about 55 kDa, and that of the other 6 chimeric proteins was about 50 kDa. Methods of SDS-PAGE and Western blotting are disclosed, for example, in patent CN101148661 B.

EXAMPLE 6

Purification of Chimeric L1 Protein and Dynamic Light Scattering Analysis (DLS) of the Particle Size

(30) Proper amount of cells expressing chimeric L1 were resuspended in 10 ml PBS. PMSF was added to a final concentration of 1 mg/ml, and cells were ruptured by ultrasonic (Ningbo Xinzhi Ultrasonic Breaker, 6# probe, 200 W, ultrasonic for 5 s, followed by an interval of 7 s, for a total duration of 10 min). The lysate was used for purification at room temperature. VLPs were depolymerized by adding 4% 13-mercaptoethanol (w/w) to the lysate, and the sample was then filtered using a 0.22 m filter followed by purifying with DMAE anion exchange chromatography or CM cation exchange chromatography (elution with 20 mM Tris, 180 mM NaCl, 4% -ME, pH 7.9), TMAE anion exchange chromatography or Q cation exchange chromatography (elution with 20 mM Tris, 180 mM NaCl, 4% -ME, pH 7.9) and hydroxyapatite chromatography (elution with 100 mM NaH.sub.2PO.sub.4, 30 mM NaCl, 4% -ME, pH 6.0), sequentially. The purified product was concentrated using a Planova ultrafiltration system and VLPs were assembled by buffer exchanging (20 mM NaH.sub.2PO.sub.4, 500 mM NaCl, pH 6.0). The above purification methods are disclosed, for example, in patents CN101293918 B, CN1976718 A and the like.

(31) The purified chimeric protein solution was subjected to DLS particle size analysis (Zetasizer Nano ZS 90 Dynamic Light Scattering Instrument, Malvern). The results are shown in Table 1, and the DLS analysis of HPV16L1CDE.sub.135-138/33dE and HPV16L1CDE.sub.135-138/33dEs was shown in FIG. 2.

(32) TABLE-US-00001 TABLE 1 DLS analysis of chimeric L1 proteins Hydrodynamic Proteins diameter (nm) PDI HPV16L1DE.sub.136-137/33dE 92.5 0.131 HPV16L1DE.sub.136-137/33dEs 98.4 0.142 HPV16L1DE.sub.135-138/33dE 91.4 0.133 HPV16L1DE.sub.135-138/33dEs 95.2 0.142 HPV16L1h4/33dE 89.4 0.176 HPV16L1h4/33dEs 82.6 0.188 HPV16L1CDE.sub.136-137/33dE 99.4 0.142 HPV16L1CDE.sub.136-137/33dEs 98.8 0.136 HPV16L1CDE.sub.135-138/33dE 91.6 0.177 HPV16L1CDE.sub.135-138/33dEs 97.9 0.143 HPV16L1Ch4/33dE 88.6 0.144 HPV16L1Ch4/33dEs 90.8 0.152

EXAMPLE 7

Observation of Chimeric VLPs by Transmission Electron Microscope (TEM)

(33) The chimeric VLPs were respectively purified using the chromatographic purification methods described in Example 6 and the dialyzed VLPs were used to prepare a copper mesh and stained with 1% uranyl acetate. After dried thoroughly, VLPs were observed with JEM-1400 electron microscope (Olympus). As shown in FIG. 3, the insect-cell-expressed chimeric cVLPs comprising L2 polypeptide in DE loop were about 50 nm in diameter, whereas cVLPs comprising L2 polypeptide in h4 region were about 35-40 nm in diameter. Diameters of Prokaryotic expressed cVLPs were consistent with that of cVLPs expressed in insect cells, cVLPs comprising L2 polypeptide in DE loop were about 50 nm in diameter, and cVLPs comprising L2 polypeptide in h4 region were about 35-40 nm in diameter. Methods of preparing copper mesh and electron microscopy are disclosed, for example, in patent CN 101148661 B.

EXAMPLE 8

Immunization of Chimeric VLPs and Detection of Neutralizing Antibody Titers

(34) Four-to-six-week old BALB/c mice were randomly divided into groups, 5 mice per group. The mice were immunized subcutaneously with 10 g of cVLP, 50 g of Al(OH).sub.3 adjuvant and 50 g of PIKA adjuvant at weeks 0, 2, 4 and 6 for 4 times. Sera were collected 2 weeks after the fourth immunization through the tail vein.

(35) Twelve types of HPV pseudoviruses were used to detect the neutralizing antibody titers of immune sera. As shown in Table 2, the results showed that cVLPs expressed in either insect cells or E. coli cells effectively induced broad-spectrum cross-neutralizing antibodies in mice, among which the immune sera of HPV16L1CDE.sub.136-137/33dE and other insect-cell-expressed cVLPs can neutralize at least 12 types of pseudoviruses. Methods for pseudovirus preparation and pseudovirus neutralization assay are disclosed, for example, in patent CN 104418942A.

(36) Moreover, chimeric proteins constructed by inserting L2 epitopes with other flexible linkers in the DE region or the h4 region included in the present invention can assemble into cVLPs. When immunizing mice using the above-mentioned strategy, these cVLPs induced comparable levels of cross-neutralizing antibodies to the cVLPs shown in Table 2. The pentamers composed of the above-mentioned 12 chimeric L1 proteins also induced cross-neutralizing antibodies in mice when using the above-mentioned immunization strategy.

(37) TABLE-US-00002 TABLE 2 Neutralizing antibody titers induced by different cVLP in mice Type Of pseudo-virus HPV16 HPV16 HPV16 HPV16 HPV16 HPV16 L1CDE.sub.136-137/ L1CDE.sub.136-137/ L1CDE.sub.135-138/ L1CDE.sub.135-138/ L1C h4/ L1C h4/ Name of Anti-sera 33dE VLP 33dEs VLP 33dE VLP 33dEs VLP 33dE VLP 33dEs VLP Mean neutralizing HPV16 128000 165800 128000 140800 211200 160400 antibody titers HPV31 25 50 50 25 25 25 HPV33 1350 2400 1325 2600 1350 1100 HPV35 100 150 112.5 100 150 100 HPV52 150 125 137.5 212.5 100 125 HPV58 1350 800 2450 1100 500 600 HPV18 250 200 300 350 150 200 HPV39 200 300 500 225 0-25 25 HPV45 300 250 400 337.5 100 125 HPV68 100 125 100 212.5 0-25 25 HPV6 500 400 600 412.5 175 150 HPV5 450 500 525 450 0-25 25