CN1578787A

CN1578787A - Vectors, constructs, and transgenic plants for hpv-11 and hpv-16 l1 capsid protein

Info

Publication number: CN1578787A
Application number: CNA028215079A
Authority: CN
Inventors: 阿尔温德·德夫希·瓦尔萨尼; 爱德华·彼得·雷比茨基; 安娜-利塞·威廉森
Original assignee: University of Cape Town
Current assignee: University of Cape Town
Priority date: 2001-08-31
Filing date: 2002-08-30
Publication date: 2005-02-09
Anticipated expiration: 2022-08-30
Also published as: WO2003018624A1; AP2004003021A0; ZA200402504B; CN1290859C

Abstract

Vectors and/or constructs of human papillomavirus genes are used in transforming host cells, such as bacteria, yeast cells, fungal cells, insect cells, mammalian cells and plant cells. The host cells are cultivated under conditions which permit spotaeousamly of at least one protein fragment of a protein encoded by the vector and/or gene construct into an immunogenic-response elicitig virus-like particle directed against human papillomavirus.

Description

About the carrier of HPV-11 and HPV-16 L1 capsid protein, construct, and transgenic plant

Background of invention

The present invention relates to carrier and/or construct, and genetically modified organism.

Following term in this manual, phrase and/or clause should be understood to be meant:

" construct "-as used herein and term are as " conjugate ", and " box " and " hybrid " synonym comprises the nucleotide sequence that directly or indirectly is connected with promotor.Construct can comprise or presentation markup, and it allows to select construct in host cell.

" expression vector "-as used herein be meant can body in or the construct of vivoexpression.

" expression "-should be understood to be meant from dna profiling by transcribing and translate production protein.

" biology of genetic modification "-as used herein is meant because with by mating or recombinate naturally or both natural modes that do not take place have been imported into the biology of nucleotide sequence as the result of human intervention.

" on silicon chip (In silico) "-part as herein described is measured and/or method can be undertaken by using suitable software for calculation.These that so carry out measure and/method should be understood to wherein involved.

" nucleotide sequence "-as used herein and term " nucleotide sequence " and/or term " Polynucleotide " and/or term " polynucleotide " synonym, and comprise genomic dna, cDNA, recombinant DNA, synthetic DNA, and RNA and above-mentioned any combination, nucleotide sequence can be no matter two strands or strand represent sense strand or antisense strand in addition.Preferably, term " nucleotide sequence " is meant DNA.

" operability connection "-as used herein being meant side by side, the expression that wherein will be encoding sequence in this manner with the promotor of encoding sequence " operability is connected " be with the matched condition of control sequence (i.e. promotor especially, enhanser and other are expressed conditioning signal) under realize being connected.

" promotor "-as used herein is the nucleotide sequence that instructs the nucleotide sequence adjustable type to transcribe.

" protein "-as used herein is to be used for representing peptide and polypeptide.

" reorganization is biological "-genetically modified organism that comprises carrier and/or express the construct of nucleotide sequence that is meant as used herein.

" genetically modified organism "-biology that comprises genetic modification as used herein, it comprises carrier and/or construct, and according to the present invention, wherein promotor can allow to express the nucleotide sequence according to carrier of the present invention and/or construct in biological, or its part.

" carrier "-expression vector that comprises as used herein, replicable vector, conversion carrier, shuttle vectors, clay, plasmid, phage, virus and yeast artificial chromosome or its any combination.

" VLP "-virus-like particle that is meant as used herein.

Papilloma virus (PVs) belongs to classification section papilloma virus subfamily (Papilomaviridae), is little, uncovered, double-stranded (ds) dna virus.Various high vertebratess of these virus infectiones and be highly species specific.After infecting, human papillomavirus (HPVs) has special tropism to undifferentiated squamous cell, remove and plantar wart, verruca plana, relevant with reproduction wart (pointed condyloma) beyond.HPV infects also relevant with the disease that is called epidermodysplasia verruciformis, and this disease is a kind of rare lifelong disease of seeing, it is characterized in that the papilloma of scattering.In addition, there has been some papilloma virus strain isolated and comprised cervical cancer, carcinoma vulvae, the epidemiology of the pernicious transfer of the urogenital cancer of penile cancer and epidermodysplasia verruciformis infringement and biological chemistry contact.In the various HPV that characterizes, be accredited as the pathogenic agent that anus is grown infection with about 27 kinds.6,11,16,18,31,35 and 42 type HPV have been the most general by evaluation, and 16,18,31,33,51 with 54 types to grow cancer relevant with anus, be considered to high-risk, more particularly HPV-16 finds in 95% cervical cancer.

Think that up to date high-risk reproduction HPVs only propagates during sexual intercourse, yet studies show that the baby obtains high-risk HPV from their mother at birth and infects.Except non-sexupara baby propagates, the investigator in deriving from maiden's Cervico-vagina sample, and detects HPVDNA in deriving from 61 vulva swabs of claiming among the asexual women who contact history of 9 in deriving from women's vaginal swab.

Initial HPV infects and causes intracutaneous tumour formation (CIN) on the neck, more generally is known as precancerous lesion.Have been found that the spontaneous regression that has CIN in some women, but this is not that such was the case with, pathology is for many years sustainable, increases the possibility to the cervical cancer progress.

From psychrotherapy, the methods of treatment of surgical excision and local therapeutic domain does not often alleviate problem, and for the patient that treatment is replied, recurrence often is a problem.As if it is infeasible finally to destroy all infected tissues, because many focuses disease is the common feature that infects with hiding.As if target design and to suppress the antiviral of virus replication invalid in the situation that HPV infects, at first as if the virus replication gene by integrating, is reset because this virus is not duplicated in keeping the cell that infects with next and disappearance and the fact that disappears.

Conventionally most of vaccine are made up of the attenuated virus of living or the virus of formalin deactivation.Owing to relate to the difficulty and the risk that produce these a large amount of conventional vaccines, the method for having emphasized to develop the viral protein subunit vaccine very much and having produced it.

Summary of the invention

According to the invention provides carrier and/or construct, it is selected from:

a.HPV11?L1?NLS ^-；

b.HPV11?L1；

c.HPV16?L1?NLS ^-；

d.HPV16?L1；

e.pART7；

f.pART27；

Particularly:

(i) HPV11 or HPV16 L1 gene (504 amino acid);

The HPV11 or the HPV16 L1 gene (NLS-, Δ 483) that (ii) lack nuclear localization signal;

The HPV11 or the HPV16 L1 gene (Δ N10) that (iii) lack 10 N-termination codons;

The HPV11 or the HPV16 L1 gene (Δ N10 Δ 483) that (iv) lack 10 N-termination codons and NLS;

(HPV11 or the HPV16 L1 gene (pen504) that v) have C to G sudden change (pen) in 428 in amino acid;

(vi) have HPV11 or HPV16 L1 gene that pen and Δ N10 modify;

(vii) have HPV11 or HPV16 L1 gene that pen and Δ 483 are modified; With

(viii) have HPV11 or HPV16 L1 gene that pen and Δ N10 Δ 483 are modified.

In addition, according to the invention provides the application in the method for transformed host cell of carrier and/or construct.

In addition, according to the invention provides with carrier and/or construct transformed host cells, wherein transformed host cells is preferably:

A. bacterium;

B. yeast cell;

C. fungal cell;

D. insect cell;

E. mammalian cell;

F. vegetable cell,

It is at least aly cultivated under the spontaneous condition that is assembled into the virus-like particle that brings out immunogenic response by carrier and/or construct encoded protein matter or proteinic fragment allowing, described virus-like particle is at human papillomavirus, more specifically human papillomavirus-11 and/or human papillomavirus-16, and most preferably carrier and/or construct are stably integrated in the genome of host cell.

Also have, according to the invention provides the inhuman many cells genetically modified organism that transforms by carrier and/or construct, it is at least aly cultivated under the spontaneous condition that is assembled into the virus-like particle that brings out immunogenic response by carrier and/or construct encoded protein matter or proteinic fragment allowing, described virus-like particle is at human papillomavirus, more specifically human papillomavirus-11 and/or human papillomavirus-16, and preferred genetically modified organism is selected from:

1. tobacco (Nicotiana tabacum) cv Xanthi;

2. tobacco cv Soulouk;

3. tomato (Lycopersicon esculentum) cv Tiny Tom; With

4. mouseearcress (Arabidopsis thaliana) cv Columbia.

In addition, the invention provides by genetically modified organism system expression protein or proteinic fragment.

The accompanying drawing summary

Feature of the present invention will become obviously from the following description of only passing through embodiment and the following stated certain embodiments of the invention with reference to the accompanying drawings, in described accompanying drawing:

Fig. 1 shows the plasmid figure of pART7;

Fig. 2 shows the plasmid figure of pART27;

The electron micrograph of the HPV-16 VLP that Fig. 3 shows is isolating from transgene tobacco, catch with the special Mab of HPV-16-; With

Fig. 4 shows the electron micrograph that belongs to isolating HPV-11 VLP (Arabidopsis) from transgenic arabidopsis.

The preferred embodiment of the invention describes in detail

The method for preparing genetically modified organism it is known to those skilled in the art that, will deeply not launch in this manual.

Clone and preparation Agrobacterium clone

Utilize HIND III and EcoR I restriction site HPV-11 L1 and HPV-16 L1 gene fragment to be cloned into separately in the multiple clone site (MCS) of initial carrier pART7 so that construct HPV11 L1 to be provided NLS ^-, HPV11 L1, HPV16 L1 NLS ^-And HPV16 L1.

By Not I digestion complete box of excision from pART7 carrier (Fig. 1), and be cloned among the binary vector pART27 (Fig. 2).

Each pART27 is cloned transfection in agrobacterium tumefaciens bacterial strain C58C1.

The leaf dish transforms and tissue culture

With Agrobacterium clone transformation of tobacco cv Xanthi and Soulouk leaf dish, on the kantlex tissue culture medium (TCM), select transformant, as (Transfection of whole plants fromwounds inoculated with Agrobacterium tumefaciens containing cDNA oftobacco mosaic virus.J.Virol.Meth.42:227-240) as described in Turpen etc.

Transform tomato cv Tiny Tom cotyledon with Agrobacterium clone, as (Transformation of tomato.J.Mol.Biol.Meth.81:359-363) as described in the Pfitzner.

The clone transforms the mouseearcress cv Columbia plant that blooms with Agrobacterium, as (Arabidopsis in the obtainable simplification of http://www.cropsci.uiuc.edu/～a-bent/protocol.hmtl transforms scheme) as described in Clough and the Bent, on the kantlex tissue culture medium (TCM), select transformant, as (Transfection of whole plants from wounds inoculated withAgrobacterium tumefaciens containing cDNA of tobacco mosaic virus.J.Virol.Meth.42:227-240) as described in Turpen etc.

The screening transgenic plant

Described methods (A plant DNA minipreparation:Version II.Cold Spring Harbour Laboratory.Cold Spring Harbour) such as use Dellaporta are extracted plant genome DNA from transformant, confirm the existence of integrator gene by PCR.

From transgenic plant, extract VLP

At 1: 2 volume (vegetable material: stir evenly the plant leaf material in damping fluid damping fluid) (PBS that uses to contain 0.5MNaCl for HPV), and on funnel, compress by cheese cloth dry until slurry.Under 4 ℃ with centrifugal 10 minutes of extract (3000rpm).10%PEG (MW8000) added supernatant liquor and allow spend the night 4 ℃ of dissolvings.Then with this centrifugal 10 minutes of 4 ℃ of following 3000rpm.Under 4 ℃ with pellet resuspended in the PBS of 1/10 initial volume (0.5M NaCl).Then under 3000rpm with centrifugal 15 minutes of mixture so that remove the insoluble component of all sex change.Extract covered on 40% the sucrose pad (in the PBS that contains 0.5M NaCl, preparing), and 100 000xg (for Sorvall SW28,24000rpm) under 10 ℃ centrifugal 2.5 hours down.Pellet resuspended is passed through 18﹠amp in a small amount of PBS (0.5M NaCl) and with suspension; The pin of 26 specifications is to reduce its viscosity.100 000xg to the linear saccharose gradient of 10-40% and under 10 ℃ (for SW28,24000rpm) rotated 3 hours with this suspension application of sample.Remove observed band under scattered light with pin and syringe, and under 4 ℃, PBS (0.5M NaCl) was dialysed 12-24 hour.

Electron microscopic analysis

VLP is trapped in bag by on the copper mesh of polyclonal antibody (specific for HPV-11 L1) R399, and with 2% uranyl acetate negative staining.Under the 200CX electron microscope, observe grid.

At R ₀In (first transgenic lines) L1 gene be incorporated in the genome of tobacco cv Xanthi and Soulouk with being stabilized.For T ₁The L1 gene of the self-pollination transgenic plant in generation (first-generation) is observed conventional Mendelian inheritances in 3: 1.To T5 (the 5th generation), detect the L1 gene at the T2 of transgenic arabidopsis dish plant (s-generation) in addition.

Observed VLP under electron microscope (with antibody capture or do not catch) is similar to those that the L1 genetic expression of using the baculovirus insect cell system produces in appearance.Observe the particulate diameter and be about 50-60nm (mouseearcress), and when checking the protein tobacco extract, observe the particle of part degraded.

Papilloma virus expression of gene in transgenic plant

In further studying, preparation comprises the transgenic lines of HPV-16 L1 gene, and it is the s-generation of selecting now at least.In addition, with HPV-11 NLS-L1 gene transformation mouseearcress and tobacco plant.Also use CRPV L1 transformation of tobacco plant recently.All these departments of botany show production VLP, although concentration is low.As if it is almost as broad as long for HPV-16 L1 VLP expression to comprise or lack the NLS sequence, although HPV-11 L1 has toxic on the plant nucleolus if navigate to.Catch HPV-16 L1 VLP by V5 MAb, as shown in Figure 3 (bar=70nm), show that they are suitable for vaccine and use on antigenicity.

(show isolating HPV-11 VLP from transgenic arabidopsis belongs in the bar=70mm) at Fig. 4.

The HPV-11 L1 albumen (NLS-that lacks nuclear localization signal, Δ 483) in transgenic plant, expresses with many different HPV-16 L1 and L1 NLS-albumen construct, these prepare the icosahedral particle VLP of T=7 after measured, be immunogenic in rabbit after injecting, as if identical on the morphology with the VLP for preparing in the baculovirus expression system at insect cell.In addition, be determined at the HPV-16 VLP monoclonal antibody (MAb V5) special for preparing in the transgenic plant and combined, and stoped the neutralizing antibody combination with conformation.These discoveries are presented at the VLP for preparing in the plant identical with the VLP for preparing on the function in insect cell.In addition, following construct by recombinant baculovirus in expressed in insect cells, its with by the special and sequence-specific Mab combination of protein bound, the identical conformation of unaltered L1:

A. the HPV16 L1 gene (Δ N10) that lacks 10 N-termination codons, it is assembled into T=1 and the icosahedron particle of T=7 in addition;

B. the HPV16 L1 gene (Δ N10 Δ 483) that lacks 10 N-termination codons and NLS, it is assembled into T=1 and the icosahedron particle of T=7 in addition;

C. have the HPV16 L1 gene (pen504) of C to G sudden change (pen) at amino acid 428 places, it is assembled into pentamer;

D. have the HPV16 L1 gene that pen and Δ N10 modify, it is assembled into pentamer;

E. have the HPV16 L1 gene that pen and Δ 483 are modified, it is assembled into pentamer; With

F. have the HPV16 L1 gene that pen and Δ N10 Δ 483 are modified, it is assembled into pentamer.

It will be apparent to those skilled in the art that these constructs can express and should work in identical mode in transgenic plant.

Only enumerated certain embodiments of the present invention although and then be apparent that this paper for those skilled in the art, other modification of the present invention and/or variant are possible.These modifications and/or variant should be considered to belong in the scope of the present invention.

Claims

1. carrier and/or gene construct, it is selected from:

a.HPV11L1NLS ^-；

b.HPV11L1；

c.HPV16L1NLS ^-；

d.HPV16L1；

E.pART7; With

f.pART27。

2. according to the carrier and/or the gene construct of claim 1, it is selected from:

(i) HPV11 or HPV16L1 gene (504 amino acid);

The HPV11 or the HPV16L1 gene (NLS-, Δ 483) that (ii) lack nuclear localization signal;

The HPV11 or the HPV16L1 gene (Δ N10) that (iii) lack 10 N-termination codons;

The HPV11 or the HPV16L1 gene (Δ N10 Δ 483) that (iv) lack 10 N-termination codons and NLS;

(HPV11 or the HPV16L1 gene (pen504) that v) have C to G sudden change (pen) in 428 in amino acid;

(i) have HPV11 or the HPV16L1 gene that pen and Δ N10 modify;

(vii) have HPV11 or HPV16L1 gene that pen and Δ 483 are modified; With

(viii) have HPV11 or HPV16L1 gene that pen and Δ N10 Δ 483 are modified.

3. claim 1 or 2 carrier and/or the application of gene construct in the method for transformed host cell.

4. carrier and/or gene construct transformed host cells with claim 1 or 2, it is selected from bacterium, yeast cell, fungal cell, insect cell, mammalian cell, and vegetable cell.

5. according to the host cell of claim 4, it is at least aly cultivated under the spontaneous condition that is assembled into the virus-like particle that brings out immunogenic response by described carrier and/or gene construct encoded protein matter or protein fragments allowing, and described virus-like particle is at human papillomavirus.

6. according to the host cell of claim 5, wherein said human papillomavirus is human papillomavirus-11 and/or human papillomavirus-16.

7. according to the host cell of claim 6, wherein said carrier and/or gene construct are stably integrated among the genome of host cell.

8. inhuman many cells genetically modified organism, it is to be transformed by the carrier of claim 1 or 2 and/or gene construct, at least aly cultivated under the spontaneous condition that is assembled into the virus-like particle that brings out immunogenic response by described carrier and/or gene construct encoded protein matter or protein fragments allowing, described virus-like particle is at human papillomavirus.

9. according to the genetically modified organism of claim 8, wherein said virus-like particle is at human papillomavirus-11 and/or human papillomavirus-16.

10. according to the genetically modified organism of claim 9, it is selected from tobacco cv Xanthi, tobacco cvSoulouk, tomato cv Tiny Tom; With mouseearcress cv Columbia.