CN101314615A - Viral capsid mosaic protein HPV16 L1-P<D>, preparation method and application thereof - Google Patents
Viral capsid mosaic protein HPV16 L1-P<D>, preparation method and application thereof Download PDFInfo
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- CN101314615A CN101314615A CNA200810069886XA CN200810069886A CN101314615A CN 101314615 A CN101314615 A CN 101314615A CN A200810069886X A CNA200810069886X A CN A200810069886XA CN 200810069886 A CN200810069886 A CN 200810069886A CN 101314615 A CN101314615 A CN 101314615A
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Abstract
The invention belongs to the gene engineering field, and discloses a capsid chimeric protein HPV16 L1-PD, the preparation method thereof and the application thereof. HPV16 L1-PD has an amino acid sequence shown as SEQ ID No.2; and the encoding gene thereof has a nucleotide sequence shown as SEQ ID No.1. The preparation method comprises the steps as follows: constituting HPV16 L1-PD gene; cloning the HPV16 L1-PD gene; constituting a recombination carrier pFastBacDual HPV16 L1-PD; preparing the recombined baculovirus containing the HPV16 L1-PD gene; expressing in insect cells; purifying HPV16 L1-PD, etc. HPV16 L1-PD can be adopted as a gene or a pharmaceutical target carrier and a vaccine transfer tool, and can be applied in preparing the therapeutic virus-like vaccine of systemic lupus erythematosus.
Description
Technical field
The present invention relates to the genetically engineered field, be specifically related to viral capsid chimeric protein HPV16 L1-P
DAnd its production and use.
Background technology
Virus-like particle (Virus-like Particles, VLPs) be a kind of on morphological structure with form of spherical particles material like the virus type, but do not contain viral genome.Basis source can be divided into and comes from virus vlps s and artificial VLPs.Mainly by the genetically engineered preparation, the latter is by artificial chemosynthesis for the former.The surface component of VLPs can carry out flexile modification transformation to satisfy different demands; Under optimum conditions, VLPs can wrap up nucleic acid, medicine or other small-molecule substance, as molecular targeted launch vehicle; VLPs also has unique immunological characteristic, can not only be by influencing antigen presenting cell performance adjuvant effect, can also design the vaccine of various ways as the integration platform of other adjuvant, peptide vaccine and nucleic acid vaccine, therefore in molecular vehicle and vaccine design, have a good application prospect.
Discover, human papillomavirus 16 types (Human Papillomavirus type-16, HPV16) main capsid protein (L1) has the characteristic that self is assembled into VLPs, and HPV16 L1VLPs can also be packaged into exogenous nucleic acid molecule the pseudo-virus (Pseudo Virus) with natural viral spline structure, can be used as the launch vehicle of gene or medicine.
Systemic lupus erythematous (systemic lupus erythematosus, SLE) be a kind of be characteristics to produce multiple autoantibody, clinical manifestation is various, can invade the autoimmune disorder of multisystem, many organs.Its etiology unknown may multiple factor be relevant with heredity, sexual hormoue, environment, infection, medicine, immunity of organism is unusual etc.Main at present application glucocorticosteroid and immunosuppressor are carried out combination therapy, but toxic side effect is obvious, immune response to body opposing pathogenic microorganism when reaction produces inhibition to abnormal immune also produces inhibition, increased the danger that the patient infects, and the invalid or weak effect of part patient treatment belongs to intractable lupus.In recent years, along with the SLE pathogenesis is further furtherd investigate, become the new direction of treatment at the selectivity targeted therapy of the key molecule of a certain link in the pathogenesis or influence morbidity and progression of disease, based on the research and development of the multiple biotechnological formulation of biotechnology and use and become the focus that treatment of autoimmune diseases is studied.
Bone-marrow-derived lymphocyte plays an important role in various autoimmune disease incidence and progress, and it not only can be used as the plasmacytic precursor that produces antibody, also can be used as the potential antigen presenting cell, the activation of helper cell; In addition, bone-marrow-derived lymphocyte also can pass through secrete cytokines, comprises IL-4, IL-6, IL-10 and IFN-γ, brings into play the immunoregulation effect, thereby becomes the emphasis of SLE targeted therapy method research in recent years.Current SLE targeted therapy strategy at the B cell has activation, the recombinant immunotoxin of interference DNA specific b cells to kill and wound pathologic B cell etc., but these schemes exist easily degraded in the body, serum half-life is short, result of treatment can not be stablized and be lasting, may cause non-specific damage behind other cytophagy, and the deficiencies such as immunne response that may bring out body.Therefore, develop biotechnological formulation and become the urgent and important work in this area with good validity, security and tolerance.But the research report of pseudo-viral vaccine of the relevant SLE therapeutic of Shang Weijian and preparation method thereof aspect up to now.
Can detect many antinuclear antibody in SLE patient's body, wherein anti-ds-DNA antibody not only has diagnostics and is worth, and is the main pathogenic antibody that triggers SLE.As the DNA specific b cells of anti-ds-DNA antibody sources, in the SLE morbidity, playing the part of pivotal player.(B cell recptor is consistent with anti-ds-DNA antibody on idiotype BCR) to the antigen receptor on DNA specific b cells surface, and they can be in conjunction with common antigen.People such as Gaynor B are affinity ligand with anti-ds-DNA antibody, and screening has obtained a plurality of epitope peptides from phage random peptide library, but analog D NA molecule and anti-ds-DNA antibodies, and one of them sequence is 10 peptides (the called after P in the present invention of DWEYSVWLSN
DPeptide), with ds-DNA antibody high-affinity (Peptide inhibition of glomerular deposition of ananti-DNA antibody.Gaynor B, et al.Proc Natl Acad Sci, 94 (5): 1955-1960,1997) is arranged.Therefore, P
DPeptide can be used to guide fusion rotein or genophore that the DNA specific b cells is killed and wounded or intervenes.
Summary of the invention
In view of this, first purpose of the present invention is to provide viral capsid chimeric protein HPV16 L1-P
D, have the aminoacid sequence shown in SEQ ID No.2.
Second purpose of the present invention is to provide viral capsid chimeric protein HPV16 L1-P
DEncoding gene, have the nucleotide sequence shown in SEQ ID No.1.
The 3rd purpose of the present invention is to provide viral capsid chimeric protein HPV16 L1-P
DThe preparation method, may further comprise the steps:
A.HPV16 L1-P
DThe structure of gene
With wild-type HPV16 L1 gene is template, adopts overlapping extension PCR method to insert coding P between 798/799 bit base of HPV16 L1 gene
DThe base sequence of peptide, design of primers is as follows: outside forward primer F is shown in SEQ ID No.3, and outside reverse primer R is shown in SEQ ID No.4, and mutagenic primer Fm is shown in SEQID No.5, and mutagenic primer Rm is shown in SEQ ID No.6; The PCR product is after agarose gel electrophoresis is identified, the purpose fragment of the about 1600bp of purifying, gained purpose fragment cloning is gone into pCR II-TOPO carrier, be transformed into DH5 α competent escherichia coli cell again, with the LB plate screening positive colony bacterium that contains penbritin, extract plasmid, adopt enzyme to cut and identify positive colony plasmid and order-checking, obtain the HPV16 L1-P of 1602bp with the PCR method
DGene has the nucleotide sequence shown in SEQ ID No.1;
B.HPV16 L1-P
DThe clone of gene
With step a gained positive colony plasmid is template, PCR method amplification HPV16 L1-P
DGene, design of primers is as follows: upstream primer I is shown in SEQ ID No.7, and downstream primer I is shown in SEQ ID No.8; The PCR product is identified once more according to the described method of step a, purifying, and the clone transforms, and screening is identified, and a series of processes such as order-checking are confirmed clone HPV16 L1-P
DThe order exactness of gene;
C. recombinant vectors pFastBacDual HPV16 L1-P
DStructure
Step b gained positive colony plasmid is carried out double digestion with EheI and XhoI, obtain HPV16 L1-P
DGene fragment is cloned into baculovirus transfer vector pFastBacDual again, obtains recombinant vectors pFastBacDual HPV16 L1-P
D
D. contain HPV16 L1-P
DThe preparation of the recombinant baculovirus of gene
According to BaculoGold
TMTransfection reagent box description operation is with the recombinant vectors pFastBacDual HPV16 L1-P of step c structure
DWith the baculovirus linear DNA be BaculoGold
TMDNA cotransfection insect cell Sf9, homologous recombination obtains to contain HPV16 L1-P in born of the same parents
DThe recombinant baculovirus of gene; Transfection Sf9 cell was cultivated 3 days for 27 ℃ in temperature, collected culture supernatant as virus stock solution used;
E. insect cell inner expression and HPV16 L1-P
DPurifying
Steps d gained virus stock solution used is infected the Sf9 cell, put temperature and cultivated 3 days for 27 ℃, collect and be subjected to transfect cell; It is among the PBS that the cell precipitation that will be contaminted is resuspended in phosphate buffered saline buffer, ultrasonic broken wall, low-speed centrifugal is collected supernatant, be transferred to and fill in the centrifuge tube that mass percentage concentration is 40% sucrose solution, the ultracentrifugation collecting precipitation, with the CsCl density gradient ultracentrifugation, buoyant density is the white protein band of 1.34g/ml in the collection tube again, promptly gets viral capsid chimeric protein HPV16 L1-P
D
The 4th purpose of the present invention is to provide viral capsid chimeric protein HPV16 L1-P
DApplication as gene or drug targeting carrier.
The 5th purpose of the present invention is to provide viral capsid chimeric protein HPV16 L1-P
DApplication as the vaccine delivery instrument.
Further, described viral capsid chimeric protein HPV16 L1-P
DApplication as SLE vaccine delivery instrument.
The 6th purpose of the present invention is to provide the SLE therapeutic pseudo-viral vaccine, and this vaccine is with viral capsid chimeric protein HPV16 L1-P
DDiphtheria toxin A chain under the parcel B cell specificity promotor Ig κ control is a DT-A gene expression profiling plasmid DNA molecule, the pseudo-virus of the simulation natural viral spline structure that self-assembly forms.
The 7th purpose of the present invention is to provide employing viral capsid chimeric protein HPV16 L1-P
DPrepare the method for the pseudo-viral vaccine of SLE therapeutic, may further comprise the steps:
A. viral capsid chimeric protein HPV16 L1-P
DPreparation
With aforementioned viral capsid chimeric protein HPV16 L1-P
DThe preparation method identical;
B. the preparation of recombinant vectors pcDNA3 Ig κ DT-A
The pTHA45 carrier contains Ig κ DT-A gene fragment, with Apa I and Bgl II double digestion pTHA45 carrier, cut glue and reclaim purifying Ig κ DT-A gene fragment, be connected with the pcDNA3 carrier that digests through same double digestion again, make recombinant vectors pcDNA3 Ig κ DT-A;
The preparation of the pseudo-viral vaccine of c.SLE therapeutic
Get step a gained viral capsid chimeric protein HPV16 L1-P
D, add denaturing agent and make protein denaturation, add step b gained recombinant vectors pcDNA3 Ig κ DT-A again, dialysed overnight in PBS makes metaprotein renaturation gradually, HPV16 L1-P in renaturation process
DParcel recombinant vectors pcDNA3 Ig κ DT-A, self-assembly forms the pseudo-viral vaccine of simulation natural viral spline structure.
Beneficial effect of the present invention is:
HPV16 L1-P of the present invention
DBe that HPV16 L1 is transformed, with P
DPeptide inserts the viral capsid chimeric protein that obtains among the HPV16 L1.This albumen adopts genetic engineering technique to be prepared, and with overlapping extension PCR method HPV16 L1 gene is made site-directed mutagenesis, obtains HPV16 L1-P
DGene is cloned into carrier pCR II-TOPO, and further directed again the insertion among the transfer vector pFastBacDual utilizes the Bac-to-Bac baculovirus expression system to express foreign protein in insect cell Sf9.RT-PCR, SDS-PAGE and Western blot identify and confirm HPV16 L1-P
DBut gene specific transcriptional and express HPV16 L1-P in the Sf9 cell
DSouthernblot identifies and shows HPV16 L1-P
DHas the desired binding ability with DNA; Transmission electron microscope is identified and is shown HPV16L1-P
DHave the characteristic that self is assembled into VLPs, and exogenous nucleic acid molecule can be packaged into pseudo-virus with natural viral spline structure.Viral capsid chimeric protein HPV16 L1-P of the present invention
DUtilization is exposed to the P on VLPs surface
DPeptide in vivo can efficient identification DNA specific b cells, thereby successful target transfection realizes genetic expression or pharmaceutically-active target in the DNA specific b cells, can be used as the launch vehicle and the vaccine delivery instrument of gene or medicine.
Adopt viral capsid chimeric protein HPV16 L1-P of the present invention
DCan prepare the pseudo-viral vaccine of SLE therapeutic and promptly simulate the nucleic acid-VLPs combination vaccine of natural viral spline structure, this vaccine and preparation method thereof has following advantage: (1) selects viral capsid chimeric protein HPV16 L1-P
DBe genophore, HPV16 L1 genetic comparison is simple, does not contain the self-replacation unit or the carcinogenic sequence of diving, and is safer than other virus vector; A little less than the immunogenicity of HPV16 L1VLPs, only trigger host weak humoral immunization and cellullar immunologic response, particularly the neutrality antibody titre of Chan Shenging is very low, can use repeatedly; HPV16 L1-P
DBut VLPs specific infection DNA specific b cells, target is strong; (2) selecting the DT-A gene expression profiling plasmid DNA molecule under the B cell specificity promotor Ig κ control is the nucleic acid molecule of VLPs parcel, express by B cell specificity promotor control toxin gene, only kill and wound pathogenic B cell clone, it is secure to kill and wound specificity; (3) the pseudo-viral surface that makes has multivalence P
DPeptide, except that with the DNA specific b cells combines, can also with the anti-ds-DNA antibodies of free in the body, help reducing the titre of pathologic autoantibody; (4) preparation process is simple, is not subjected to strict P3 Biosafety condition restriction, and product has better susceptibility and specificity; (5) vaccine of the present invention comes from artificial design, structure fully, and convenient further the modification transformed.
With MRL/lpr lupus mouse is animal model, estimate the result of treatment of the pseudo-viral vaccine of SLE therapeutic of the present invention by the mode of tail vein feedback, found that the urine protein after treatment group mouse is treated, serum urea nitrogen, creatinine and anti-ds-DNA antibody all descend, with the treatment before compared significant difference, and there was no significant difference before and after the treatment of control group, and the mean survival time (MST) of treatment group mouse, is than the obvious prolongation of control group, show that the pseudo-viral vaccine of SLE therapeutic of the present invention can significantly improve immune state and the renal function of MRL/lpr mouse, improve the mean survival time (MST) of mouse, has good potential applicability in clinical practice, can be the patient especially the patient of those traditional immunosuppressant therapy poor effect novel target biology preparation is provided, also the medicament research and development for autoimmune disorders such as SLE provides new mentality of designing.
Other advantages of the present invention, target, to set forth in the following description to a certain extent with feature, and to a certain extent,, perhaps can obtain instruction from the practice of the present invention based on being conspicuous to those skilled in the art to investigating hereinafter.Target of the present invention and other advantages can realize and obtain by following specification sheets and claims.
Description of drawings
In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is described in further detail below in conjunction with accompanying drawing, wherein:
Fig. 1 is HPV16 L1-P
DThe agarose gel electrophoresis of the PCR product of gene is identified figure;
Fig. 2 is recombinant cloning vector pCR II HPV16 L1-P
DEnzyme cut the agarose gel electrophoresis evaluation figure of product;
Fig. 3 is viral capsid chimeric protein HPV16 L1-P
DSDS-PAGE and Western-blot identify figure;
Fig. 4 is viral capsid chimeric protein HPV16 L1-P
DTransmission electron microscope picture with the pseudo-viral vaccine of SLE therapeutic;
Fig. 5 is two groups of survival rate comparison diagrams after the mouse treatment.
Embodiment
Hereinafter with reference to accompanying drawing, the preferred embodiments of the present invention are described in detail.Should be appreciated that preferred embodiment only for the present invention is described, rather than in order to limit protection scope of the present invention.The experimental technique of unreceipted actual conditions in the preferred embodiment, usually according to normal condition, the molecular cloning experiment guide (third edition for example, J. work such as Sa nurse Brooker, Huang Peitang etc. translate, Science Press, 2002) described in condition, or the condition of advising according to manufacturer.
(1) viral capsid chimeric protein HPV16 L1-P
DPreparation
1.HPV16 L1-P
DThe structure of gene
(Overlap-extension PCR, the OE-PCR) principle of site-directed mutagenesis design and synthesize 4 primers: outside forward primer F is shown in SEQ ID No.3, and underscore partly is a Bgl II restriction enzyme site according to overlapping extension PCR method; Outside reverse primer R is shown in SEQ ID No.4; Mutagenic primer Fm is shown in SEQ ID No.5, and underscore partly is the mutational site; Mutagenic primer Rm is shown in SEQ ID No.6, and underscore partly is the mutational site;
Get human cervical carcinoma cell strain Caski, add Tripure reagent, extract cell total rna according to the reagent explanation;
Reverse transcription: the total RNA with extraction is a template, with Oligo (dT)
16For primer carries out reverse transcription, obtain wild-type HPV16 L1 DNA, concrete grammar is: getting concentration is that total RNA 5 μ l of 100 μ g/ml, primer 1 μ l, the distilled water that concentration is 100 μ g/ml replenish volume to 10 μ l, 70 ℃ of water-baths 5 minutes, ice bath is 5 minutes immediately, dNTP mixing solutions 2 μ l, the RNA enzyme arrestin 20U, the distilled water that add 10 * reversed transcriptive enzyme damping fluid, 2 μ l, MMLV reversed transcriptive enzyme 20U, concentration again and be 10mmol/L replenish volume to 20 μ l, 42 ℃ of water-baths 1 hour, 95 ℃ of sex change 5 minutes;
First round PCR: with wild-type HPV16 L1 DNA is template, is the upstream and downstream primer with outside forward primer F and mutagenic primer Rm, carries out first round pcr amplification, obtains to contain the dna fragmentation FRm of mutational site and upstream sequence thereof;
Second takes turns PCR: with wild-type HPV16 L1 DNA is template, is the upstream and downstream primer with mutagenic primer Fm and outside reverse primer R, carries out second and takes turns pcr amplification, obtains to contain the dna fragmentation FmR of mutational site and downstream sequence thereof;
Third round PCR: with first and second 2 partly overlapping dna fragmentation FRm, FmR that take turns the PCR acquisition is template, with outside forward primer F and outside reverse primer R is the upstream and downstream primer, carry out the third round pcr amplification, the total length that acquisition is spliced by dna fragmentation FRm and FmR contains the dna fragmentation FR in mutational site, i.e. HPV16 L1-P
DGene;
The reaction system of above three-wheel PCR is identical with reaction conditions, and reaction system is: 10 * PCR reaction buffer, 5 μ l, concentration are that the dNTP mixing solutions 1 μ l of 10mmol/L, each 0.4 μ l of upstream and downstream primer, template 2 μ l, Taq archaeal dna polymerase 2U, the concentration that concentration is 10 μ mol/L are the MgCl of 50mmol/L
2Solution 1 μ l, distilled water replenish volume to 50 μ l; Reaction conditions is: 94 ℃ of pre-sex change 5 minutes, and 1 minute, 45 ℃ annealing of 94 ℃ of sex change were extended 2 minutes for 30 seconds, 72 ℃ then, totally 30 circulations, last 72 ℃ were extended 4 minutes;
It is that 1% agarose gel electrophoresis is identified that third round PCR product adopts mass percentage concentration, the result as shown in Figure 1, wherein 1 swimming lane is the PCR molecular weight standard, 2 swimming lanes are the PCR product, from figure as can be known, a specific DNA band occurs, conform to the theoretical prediction value in the position of about 1600bp;
Adopt gel recovery test kit (worker's biotechnology company limited is given birth in Shanghai) to carry out purifying with being accredited as correct third round PCR product, according to the test kit description operation, select the target DNA fragment of about 1600bp to cut glue recovery purifying, obtain HPV16 L1-P
DGene;
With HPV16 L1-P
DGene adds Taq archaeal dna polymerase and dATP solution, with 72 ℃ of temperature, carried out PCR in 30 minutes, makes HPV16 L1-P
DGene 3 ' end adds " a " base, agarose gel electrophoresis is identified the PCR product, cut glue and reclaim the purifying target DNA fragment, the gained target DNA fragment is connected with pCR II-TOPO carrier (sigma company), method of attachment is: concentration is that the target DNA fragment 5 μ l of 100 μ g/ml, the carrier 3 μ l that concentration is 100 μ g/ml, T4DNA ligase enzyme 0.5 μ l, 10 * damping fluid, 1 μ l, the distilled water that concentration is 100 μ g/ml replenish volume to 10 μ l, put 16 ℃ of temperature and hatched 8 hours, the directed recombinant cloning vector pCR II HPV16 L1-P that makes up
D
With recombinant cloning vector pCR II HPV16 L1-P
DBe transformed into CaCl
2The DH5 α competent escherichia coli cell of method preparation, method for transformation is: with concentration is that to join cell density be 1 * 10 for the recombinant cloning vector 2 μ l of 100 μ g/ml
6Among the competent cell 200 μ l of/ml, ice bath 30 minutes, 42 ℃ of water-bath heat-shockeds 90 seconds, ice bath is 2 minutes immediately, adds liquid LB substratum again, and in 37 ℃ of temperature, 150r/min jolting 1 hour makes transformant;
Getting transformant coats on the LB flat board that contains penbritin, being 37 ℃ in temperature was just putting 1 hour, inversion is spent the night, 3 white colonies of picking at random from the LB flat board, be inoculated in the liquid LB substratum, in 37 ℃ of temperature, the 150r/min jolting is spent the night, adopt plasmid extraction kit (omega company) to extract plasmid in a small amount, cut and PCR method evaluation positive colony plasmid with enzyme, enzyme cutting method is: concentration is the plasmid 10 μ l of 100 μ g/ml, concentration is the Bgl II restriction enzyme 1 μ l of 100 μ g/ml, 10 * damping fluid, 3 μ l, distilled water replenishes volume to 30 μ l, putting 37 ℃ of temperature hatched 4 hours, enzyme is cut product and is identified with agarose gel electrophoresis, the result as shown in Figure 2, wherein 1 swimming lane is a dna molecular amount standard, and 2 swimming lanes are that enzyme is cut product, from scheming visible two DNA band, a treaty 1600bp wherein is with theoretical prediction value basically identical; The PCR method is: to cut the plasmid that is accredited as positive colony through enzyme is template, with the described outside of step 1.1 forward primer F and outside reverse primer R is the upstream and downstream primer, carry out pcr amplification with described PCR reaction system of step 1.2 and condition, agarose gel electrophoresis is identified the PCR product, obtain the band of about 1600bp, consistent with the electrophoretic band of step 1.2 gained third round PCR product; Get the positive colony plasmid, entrust Shanghai to give birth to worker's biotechnology company and measure the insertion fragments sequence, obtain the dna sequence dna of 1602bp, shown in SEQ ID No.1, with HPV16 L1-P
DThe theoretical prediction value unanimity of gene;
2.HPV16 L1-P
DThe clone of gene
According to HPV16 L1-P
DGene order, in conjunction with the restriction enzyme site on the baculovirus transfer vector pFastBacDual, design following primer: upstream primer I is shown in SEQ ID No.7, and underscore partly is the EheI restriction enzyme site; Downstream primer I is shown in SEQ ID No.8, and underscore partly is the XhoI restriction enzyme site;
With step 1 gained positive colony plasmid is template, adopt the upstream and downstream primer I, carry out pcr amplification with described PCR reaction system of step 1 and condition, agarose gel electrophoresis is identified the PCR product, cut glue and reclaim the purifying target DNA fragment, again according to the described method of step 1 clone, screening positive clone plasmid and carry out sequencing, confirm clone HPV16 L1-P
DThe order exactness of gene;
3. recombinant vectors pFastBacDual HPV16 L1-P
DStructure
The positive colony plasmid that step 2 is identified through order-checking carries out double digestion with EheI and XhoI, and agarose gel electrophoresis is identified the double digestion product, cuts the target DNA fragment that glue reclaims the about 1600bp of purifying; With the gained target DNA fragment be connected the directed recombinant vectors pFastBacDual HPV16L1-P that makes up with the postdigestive baculovirus transfer vector pFastBacDual of XhoI double digestion (sigma company) through EheI by same procedure
D
With gained recombinant vectors pFastBacDual HPV16 L1-P
DBe transformed into CaCl
2The DH5 α competent escherichia coli cell of method preparation, screening positive clone plasmid also identify through order-checking, and the result shows that plasmid that experiment obtains is for containing HPV16 L1-P
DThe recombinant plasmid of gene;
4. contain HPV16 L1-P
DThe preparation of the recombinant baculovirus of gene
It is in 10% the FCS substratum that the Sf9 cell is put mass percentage concentration, maintains growth under 27 ℃ of conditions of temperature, when treating that cell grows to the 50-70% individual layer in culture dish, according to BaculoGold
TMTransfection reagent box (sigma company) description operation is with the recombinant vectors pFastBacDual HPV16 L1-P of step 3 structure
DWith baculovirus linear DNA (BaculoGold
TMDNA) cotransfection makes it in the Sf9 cell homologous recombination and obtains to contain HPV16 L1-P
DThe recombinant baculovirus of gene; Transfection Sf9 cell was cultivated 3 days for 27 ℃ in temperature, and the cytopathy variability reaches more than 90%, and visible cell nuclear increases, and recombinate shape virus infection features such as kytoplasm muddiness, cell rounding are collected culture supernatant as virus stock solution used;
5. insect cell inner expression and HPV16 L1-P
DPurifying
Is the Sf9 cell of 5 infection logarithmic phases with step 4 gained virus stock solution used with infection multiplicity (MOI), puts 27 ℃ of cultivations of temperature, and obvious pathology can appear in cell after 3 days, collects and is subjected to transfect cell; The concentration that the cell precipitation that is contaminted is resuspended in precooling is that 0.1mol/L, pH value are among 7.4 the PBS, ultrasonic broken wall, low-speed centrifugal is collected supernatant, be transferred to and fill in the centrifuge tube that mass percentage concentration is 40% sucrose solution, the ultracentrifugation collecting precipitation, with the CsCl density gradient ultracentrifugation, buoyant density is the white protein band of 1.34g/ml in the collection tube again, promptly gets viral capsid chimeric protein HPV16 L1-P
D, its aminoacid sequence is shown in SEQ ID No.2.
(2) viral capsid chimeric protein HPV16 L1-P
DEvaluation
1.RT-PCR identify HPV16 L1-P
DTranscribe situation in the born of the same parents of gene
Get the Sf9 cell that is contaminted of collection, extract total RNA, adopt RT-PCR test kit (TaKaRa company) to carry out RT-PCR again, agarose gel electrophoresis is identified the PCR product, and the result has an obvious band at about 1600bp place, match with theoretic product length; Cut glue and reclaim purified pcr product and carry out dna sequence analysis, confirm HPV16 L1-P
DGene is transcribed at Sf9 cell internal specific.
2.SDS-PAGE identify HPV16 L1-P with Western blot
DExpression amount and specificity
Get the HPV16 L1-P that makes
D, the functional quality percentage concentration is that 5% concentrated glue, mass percentage concentration are that 12.5% separation gel carries out SDS-PAGE, after gel electrophoresis finishes, glue is cut, half carries out the coomassie brilliant blue staining analysis; Second half transfer printing pvdf membrane is washed and is added an anti-mouse-anti people HPV16 L1-P behind the membrane closure
D, put 37 ℃ of temperature and hatched 1 hour, wash that to add sheep anti mouse two behind the film anti-, put 37 ℃ of temperature and hatched 1 hour, develop the color after washing film, carry out Western blot and analyze.
SDS-PAGE result as shown in Figure 3A, the M swimming lane is a protein molecular weight standard among the figure, the 1-4 swimming lane is HPV16 L1-P
D, it is that the specific proteins of 63kD is expressed that the Sf9 cell that as seen infects recombinant baculovirus has a part amount, i.e. HPV16 L1-P
DSpecific expressed; The Xylene Brilliant Cyanine G method records 500ml and is contaminted and contains HPV16 L1-P in the Sf9 cell
D0.5mg, the HPV16 L1-P of 1-3 swimming lane wherein
DConcentration is determined as 209,263,82 μ g/ml respectively, 4 swimming lane undetermined HPV16 L1-P
DConcentration.
Western blot result is shown in Fig. 3 B, and wherein the 1-4 swimming lane is HPV16 L1-P
D(identical with Fig. 3 A) as seen having a band with the corresponding 63kD of SDS-PAGE, show the Sf9 cell expressing that infects recombinant baculovirus with anti-HPV16 L1-P
DAntibodies specific bonded albumen, i.e. HPV16 L1-P
D
3.Southern blot identifies HPV16 L1-P
DBonding force with DNA
With the HPV16 L1-P that makes
DSeparate by agarose gel electrophoresis, then will its sex change cracking, hybridize with the dna probe of digoxigenin labeled after changeing film, renaturation, develop the color after washing film, observe.The result is presented at special position and the colour developing band occurs, shows HPV16 L1-P
DHas the desired binding ability with DNA.
4. transmission electron microscope is identified HPV16 L1-P
DSpontaneous assemble ability
With the HPV16 L1-P that makes
DDripping on 200 order copper mesh, is that 1% water-soluble acetic acid uranium carries out negative staining, observation HPV16 L1-P under the 80kV transmission electron microscope with mass percentage concentration
DForm, the result shown in Fig. 4 A, HPV16L1-P
DRounded granular structure, diameter is about 50nm, shows under other viral protein situation of shortage HPV16 L1-P
DCan spontaneous formation VLPs.
(3) preparation of the pseudo-viral vaccine of SLE therapeutic
1. viral capsid chimeric protein HPV16 L1-P
DPreparation
Be prepared according to (one) described method;
2. the preparation of recombinant vectors pcDNA3 Ig κ DT-A
PTHA45 carrier (sigma company) contains Ig κ DT-A gene fragment, this carrier is behind Apa I and Bgl II double digestion, the Ig κ DT-A gene fragment of inserting is through agarose gel electrophoresis separation, recovery, purifying, be connected under the effect of T4 ligase enzyme with the pcDNA3 carrier (sigma company) that digests through same double digestion again, obtain pcDNA3 Ig κ DT-A recombinant vectors with CMV promotor among the Ig κ DT-A gene fragment replacement fabric shuttle-type plasmid pcDNA3;
3.SLE the preparation of the pseudo-viral vaccine of therapeutic
With step 1 gained viral capsid chimeric protein HPV16 L1-P
DWith concentration is that 0.1mol/L, pH value are that 7.4 PBS dissolves, make the protein solution that concentration is 1mg/ml, adding 2 mercapto ethanol to final volume percentage concentration again is 5%, put 4 ℃ of reactions of temperature and carried out protein denaturation in 16 hours, in the protein solution of sex change, add step 2 gained recombinant vectors pcDNA3 Ig κ DT-A 2mg then, be transferred in the dialysis tubing behind the mixing, in concentration is that 0.1mol/L, pH value are in 4 ℃ of dialysed overnight of temperature among 7.4 the PBS, make metaprotein renaturation gradually, HPV16 L1-P in renaturation process
DParcel recombinant vectors pcDNA3 Ig κ DT-A, self-assembly forms the pseudo-viral vaccine of SLE therapeutic.
(4) evaluation of the pseudo-viral vaccine of SLE therapeutic
Adopt the structure of the pseudo-viral vaccine of transmission electron microscope observing gained SLE therapeutic, transmission electron microscope picture as shown in Figure 4, A is HPV16 L1-P among the figure
DStoste, B is the HPV16 L1-P after the sex change
D, C is the pseudo-viral vaccine of SLE therapeutic, from figure as can be known, and HPV16 L1-P
DRounded granular structure, it is dissociated into tiny molecular structure under the effect of denaturing agent, but can be in PBS renaturation gradually, recover original granular structure again.
(5) experimental study of the pseudo-viral vaccine therapy MRL/lpr mouse systemic lupus erythematosus of SLE therapeutic
One, experimental technique
1. animal grouping and treatment: the MRL/lpr lupus mouse 12 (being provided by Chinese Academy of Sciences's Shanghai Experimental Animal Center) of 3 monthly ages, body weight 20-30g is divided into two groups at random: experimental group and control group, 6 every group; Experimental group feeds back the pseudo-viral vaccine of SLE therapeutic of the present invention in the tail vein, and 1 time weekly, each 100 μ l feed back 3 times continuously; Control group is handled with method, but is that 0.1mol/L, pH value are that 7.4 PBS replaces the pseudo-viral vaccine of SLE therapeutic with isopyknic concentration; Respectively at before the treatment and after the treatment, in the blood sampling of mouse vena orbitalis posterior clump, separation of serum is standby.
2. the mensuration of urine protein: respectively at before the treatment and after the treatment, adopt metabolic cage accurately to measure mouse twenty-four-hour urine amount, measure urine albumen amount with the Xylene Brilliant Cyanine G method.
3. the mensuration of serum urea nitrogen, creatinine: get mice serum 100 μ l, measure blood urea nitrogen, creatinine with automatic biochemical analyzer.
4. the mensuration of anti-ds-DNA antibody titers: adopt enzyme-linked immunosorbent assay (ELISA) to measure, concrete grammar is: adding concentration in enzyme plate is the salmon sperm dna solution of 100 μ g/ml, 100 μ l/ holes, bag was by 10 hours under room temperature, with concentration is 0.1mol/L, the pH value is 7.4 PBS washing 3 times, the adding mass percentage concentration is 1% bovine serum albumin (BSA) solution, 200 μ l/ holes, sealing is 2 hours under room temperature, PBS washing 3 times, add mice serum by dilution in 1: 100,100 μ l/ holes are put 37 ℃ of temperature and were hatched 2 hours, PBS washing 3 times, adding concentration is that the goat-anti mouse two of horseradish peroxidase (HRP) mark of 100 μ l/ml resists, 100 μ l/ holes are put 37 ℃ of temperature and were hatched 1 hour, PBS washing 3 times, add the substrate solution colour developing, measure absorbancy at wavelength 492nm place with microplate reader.
5. the mensuration of mouse survival rate: the existence situation in January to April of observing two groups of mouse after treatment.
Two, experimental result
1. the mensuration of urine protein
The twenty-four-hour urine protein content sees Table 1 before and after two groups of mouse treatments, and the urine albumen amount after experimental mice is treated is than obviously reduction (P＜0.05) before treating, and the urine albumen amount of control group mice treatment front and back does not have notable difference (P＞0.05).
Twenty-four-hour urine protein content before and after the table 1. liang group mouse treatment (mg/24h, x ± s)
2. the mensuration of serum urea nitrogen, creatinine
Serum urea nitrogen, creatinine amount see Table 2 before and after two groups of mouse treatments, serum urea nitrogen after the experimental mice treatment, creatinine amount obviously reduce (P＜0.05) than treatment is preceding, and serum urea nitrogen, creatinine amount before and after the control group mice treatment do not have notable difference (P＞0.05).
Serum urea nitrogen before and after the treatment of table 2. a liang group mouse, creatinine amount (μ mol/L, x ± s)
3. the mensuration of anti-ds-DNA antibody titers
Anti-ds-DNA antibody titers sees Table 3 before and after two groups of mouse treatments, anti-ds-DNA antibody titers after the experimental mice treatment obviously reduces (P＜0.05) than treatment is preceding, and the anti-ds-DNA antibody titers before and after the control group mice treatment does not have notable difference (P＞0.05).
Anti-ds-DNA antibody titers before and after the table 3. liang group mouse treatment (the OD value, x ± s)
4. the mensuration of mouse survival rate
Survival rate comparison diagram after two groups of mouse are treated is seen Fig. 5, as shown in Figure 5, treatment back 1st month, the survival rate of two groups of mouse does not have notable difference (P＞0.05), but the survival rate obvious difference of two groups of mouse (P＜0.05) after 2nd month, the survival rate of experimental mice is apparently higher than control group.
Three, experiment conclusion
The pseudo-viral vaccine of New type of S LE therapeutic of the present invention can significantly improve immune state and the renal function of MRL/lpr mouse, improves the mouse mean survival time (MST).
Although by reference some preferred embodiment of the present invention, invention has been described, but those of ordinary skill in the art is to be understood that, can make various changes to it in the form and details, and the spirit and scope of the present invention that do not depart from appended claims and limited.
Sequence table
＜110〉Military Medical Univ No.3, P.L.A
＜120〉viral capsid chimeric protein HPV16 L1-P
DAnd its production and use
<160>8
<210>1
<211>1602
<212>DNA
＜213〉artificial sequence
<220>
<221>CDS
<222>(1)...(1602)
<220>
＜223〉description of artificial sequence: the HPV16 L1-P that HPV16 L1 gene site-directed mutagenesis is obtained
DGene
<400>1
tgt?cgt?act?acc?atc?acc?atc?acc?atc?acg?att?acg?ata?tcc?caa?cga 48
Met?Ser?Tyr?Tyr?His?His?His?His?His?His?Asp?Tyr?Asp?Ile?Pro?Thr
1 5 10 15
ccg?aaa?acc?tgt?att?ttc?agg?gcg?cct?ctc?ttt?ggc?tgc?cta?gtg?agg 96
Thr?Glu?Asn?Leu?Tyr?Phe?Gln?Gly?Ala?Ser?Leu?Trp?Leu?Pro?Ser?Glu
20 25 30
cca?ctg?tct?act?tgc?ctc?ctg?tcc?cag?tat?cta?agg?ttg?taa?gca?cgg?144
Ala?Thr?Val?Tyr?Leu?Pro?Pro?Val?Pro?Val?Ser?Lys?Val?Val?Ser?Thr
35 40 45
atg?aat?atg?ttg?cac?gca?caa?aca?tat?att?atc?atg?cag?gaa?cat?cca?192
Asp?Glu?Tyr?Val?Ala?Arg?Thr?Asn?Ile?Tyr?Tyr?His?Ala?Gly?Thr?Ser
50 55 60
gac?tac?ttg?cag?ttg?gac?atc?cct?att?ttc?cta?tta?aaa?aac?cta?aca?240
Arg?Leu?Leu?Ala?Val?Gly?His?Pro?Tyr?Phe?Pro?Ile?Lys?Lys?Pro?Asn
65 70 75 80
ata?aca?aag?tat?tag?ttc?cta?aag?tat?cag?gat?tac?aat?aca?ggg?tat 288
Asn?Asn?Lys?Val?Leu?Val?Pro?Lys?Val?Ser?Gly?Leu?Gln?Tyr?Arg?Val
85 90 95
tta?gaa?tac?att?tac?ctg?acc?cca?ata?agt?ttg?gtt?ttc?ctg?aca?cct 336
Phe?Arg?Ile?His?Leu?Pro?Asp?Pro?Asn?Lys?Phe?Gly?Phe?Pro?Asp?Thr
100 105 110
cat?ttt?ata?atc?cag?ata?cac?agc?ggc?tgg?ttt?ggg?cct?gtg?tag?gtg 384
Ser?Phe?Tyr?Asn?Pro?Asp?Thr?Gln?Arg?Leu?Val?Trp?Ala?Cys?Val?Gly
115 120 125
ttg?agg?tag?gtc?gtg?gtc?agc?cat?tag?gtg?tgg?gca?tta?gtg?gcc?atc 432
Val?Glu?Val?Gly?Arg?Gly?Gln?Pro?Leu?Gly?Val?Gly?Ile?Ser?Gly?His
130 135 140
ctt?tat?taa?ata?aat?tgg?atg?aca?cag?aaa?atg?cta?gtg?ctt?atg?cag 480
Pro?Leu?Leu?Asn?Lys?Leu?Asp?Asp?Thr?Glu?Asn?Ala?Ser?Ala?Tyr?Ala
145 150 155 160
caa?atg?cag?gtg?tgg?ata?ata?gag?aat?gta?tat?cta?tgg?att?aca?aac 528
Ala?Asn?Ala?Gly?Val?Asp?Asn?Arg?Glu?Cys?Ile?Ser?Met?Asp?Tyr?Lys
165 170 175
aaa?cac?aat?tgt?gtt?taa?ttg?gtt?gca?aac?cac?cta?tag?ggg?aac?act 576
Gln?Thr?Gln?Leu?Cys?Leu?Ile?Gly?Cys?Lys?Pro?Pro?Ile?Gly?Glu?His
180 185 190
ggg?gca?aag?gat?ccc?cat?gta?cca?atg?ttg?cag?taa?atc?cag?gtg?att 624
Trp?Gly?Lys?Gly?Ser?Pro?Cys?Thr?Asn?Val?Ala?Val?Asn?Pro?Gly?Asp
195 200 205
gtc?cac?cat?tag?agt?taa?taa?aca?cag?tta?ttc?agg?atg?gtg?ata?tgg 672
Cys?Pro?Pro?Leu?Glu?Leu?Ile?Asn?Thr?Val?Ile?Gln?Asp?Gly?Asp?Met
210 215 220
ttg?ata?ctg?gct?ttg?gtg?cta?tgg?act?tta?cta?cat?tac?agg?cta?aca 720
Val?Asp?Thr?Gly?Phe?Gly?Ala?Met?Asp?Phe?Thr?Thr?Leu?Gln?Ala?Asn
225 230 235 240
aaa?gtg?aag?ttc?cac?tgg?ata?ttt?gta?cat?cta?ttt?gca?aat?atc?cag 768
Lys?Ser?Glu?Val?Pro?Leu?Asp?Ile?Cys?Thr?Ser?Ile?Cys?Lys?Tyr?Pro
245 250 255
att?ata?tta?aaa?tgg?tgt?cag?aac?cat?atg?gcg?aca?gct?tat?ttt?ttt 816
Asp?Tyr?Ile?Lys?Met?Val?Ser?Glu?Pro?Tyr?Gly?Asp?Ser?Leu?Phe?Phe
260 265 270
att?tac?gaa?ggg?aac?aaa?tgt?ttg?tta?gac?att?tat?tta?ata?ggg?ctg 864
Tyr?Leu?Arg?Arg?Glu?Gln?Met?Phe?Val?Arg?His?Leu?Phe?Asn?Arg?Ala
275 280 285
gtg?ctg?act?ggg?agt?act?cgg?ttg?gtg?aaa?atg?tac?cag?acg?att?tat 912
Gly?Ala?Asp?Trp?Glu?Tyr?Ser?Val?Gly?Glu?Asn?Val?Pro?Asp?Asp?Leu
290 295 300
aca?tta?aag?gct?ctg?ggt?cta?ctg?caa?att?tag?cca?gtt?cga?att?att 960
Tyr?Ile?Lys?Gly?Ser?Gly?Ser?Thr?Ala?Asn?Leu?Ala?Ser?Ser?Asn?Tyr
305 310 315 320
ttc?cta?cac?cta?gtg?gtt?cta?tgg?tta?cct?ctg?atg?ccc?aaa?tat?tca 1008
Phe?Pro?Thr?Pro?Ser?Gly?Ser?Met?Val?Thr?Ser?Asp?Ala?Gln?Ile?Phe
325 330 335
ata?aac?ctt?att?ggt?tac?aac?gag?cac?agg?gcc?aca?ata?atg?gca?ttt 1056
Asn?Lys?Pro?Tyr?Trp?Leu?Gln?Arg?Ala?Gln?Gly?His?Asn?Asn?Gly?Ile
340 345 350
gtt?ggg?gta?acc?aac?tat?ttg?tta?ctg?ttg?ttg?ata?cta?cac?gca?gta 1104
Cys?Trp?Gly?Asn?Gln?Leu?Phe?Val?Thr?Val?Val?Asp?Thr?Thr?Arg?Ser
355 360 365
caa?ata?tgt?cat?tat?gtg?ctg?cca?tat?cta?ctt?cag?aaa?cta?cat?ata 1152
Thr?Asn?Met?Ser?Leu?Cys?Ala?Ala?Ile?Ser?Thr?Ser?Glu?Thr?Thr?Tyr
370 375 380
aaa?ata?cta?act?tta?agg?agt?acc?tac?gac?atg?ggg?agg?aat?atg?att 1200
Lys?Asn?Thr?Asn?Phe?Lys?Glu?Tyr?Leu?Arg?His?Gly?Glu?Glu?Tyr?Asp
385 390 395 400
tac?agt?tta?ttt?ttc?aac?tgt?gca?aaa?taa?cct?taa?ctg?cag?acg?tta 1248
Leu?Gln?Phe?Ile?Phe?Gln?Leu?Cys?Lys?Ile?Thr?Leu?Thr?Ala?Asp?Val
405 410 415
tga?cat?aca?tac?att?cta?tga?att?cca?cta?ttt?tgg?agg?act?gga?att 1296
Met?Thr?Tyr?Ile?His?Ser?Met?Asn?Ser?Thr?Ile?Leu?Glu?Asp?Trp?Asn
420 425 430
ttg?gtc?tac?aac?ccc?ccc?cag?gag?gca?cac?tag?aag?ata?ctt?ata?ggt 1344
Phe?Gly?Leu?Gln?Pro?Pro?Pro?Gly?Gly?Thr?Leu?Glu?Asp?Thr?Tyr?Arg
435 440 445
ttg?taa?cat?ccc?agg?caa?ttg?ctt?gtc?aaa?gac?ata?cac?ctc?cag?cac 1392
Phe?Val?Thr?Ser?Gln?Ala?Ile?Ala?Cys?Gln?Arg?His?Thr?Pro?Pro?Ala
450 455 460
cta?aag?aag?atc?ccc?tta?aaa?aat?aca?ctt?ttt?ggg?aag?taa?att?taa 1440
Pro?Lys?Glu?Asp?Pro?Leu?Lys?Lys?Tyr?Thr?Phe?Trp?Glu?Val?Asn?Leu
465 470 475 480
agg?aaa?agt?ttt?ctg?cag?acc?tag?atc?agt?ttc?ctt?tag?gac?gca?aat 1488
Lys?Glu?Lys?Phe?Ser?Ala?Asp?Leu?Asp?Gln?Phe?Pro?Leu?Gly?Arg?Lys
485 490 495
ttt?tac?tac?aag?cag?gat?tga?agg?cca?aac?caa?aat?tta?cat?tag?gaa 1536
Phe?Leu?Leu?Gln?Ala?Gly?Leu?Lys?Ala?Lys?Pro?Lys?Phe?Thr?Leu?Gly
500 505 510
aac?gaa?aag?cta?cac?cca?cca?cct?cat?cta?cct?cta?caa?ctg?cta?aac 1584
Lys?Arg?Lys?Ala?Thr?Pro?Thr?Thr?Ser?Ser?Thr?Ser?Thr?Thr?Ala?Lys
515 520 525
gca?aaa?aac?gta?agc?tgt 1602
Arg?Lys?Lys?Arg?Lys?Leu
530
<210>2
<211>534
<212>PRT
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: viral capsid chimeric protein HPV16 L1-P
D
<400>2
Met?Ser?Tyr?Tyr?His?His?His?His?His?His?Asp?Tyr?Asp?Ile?Pro?Thr
1 5 10 15
Thr?Glu?Asn?Leu?Tyr?Phe?Gln?Gly?Ala?Ser?Leu?Trp?Leu?Pro?Ser?Glu
20 25 30
Ala?Thr?Val?Tyr?Leu?Pro?Pro?Val?Pro?Val?Ser?Lys?Val?Val?Ser?Thr
35 40 45
Asp?Glu?Tyr?Val?Ala?Arg?Thr?Asn?Ile?Tyr?Tyr?His?Ala?Gly?Thr?Ser
50 55 60
Arg?Leu?Leu?Ala?Val?Gly?His?Pro?Tyr?Phe?Pro?Ile?Lys?Lys?Pro?Asn
65 70 75 80
Asn?Asn?Lys?Val?Leu?Val?Pro?Lys?Val?Ser?Gly?Leu?Gln?Tyr?Arg?Val
85 90 95
Phe?Arg?Ile?His?Leu?Pro?Asp?Pro?Asn?Lys?Phe?Gly?Phe?Pro?Asp?Thr
100 105 110
Ser?Phe?Tyr?Asn?Pro?Asp?Thr?Gln?Arg?Leu?Val?Trp?Ala?Cys?Val?Gly
115 120 125
Val?Glu?Val?Gly?Arg?Gly?Gln?Pro?Leu?Gly?Val?Gly?Ile?Ser?Gly?His
130 135 140
Pro?Leu?Leu?Asn?Lys?Leu?Asp?Asp?Thr?Glu?Asn?Ala?Ser?Ala?Tyr?Ala
145 150 155 160
Ala?Asn?Ala?Gly?Val?Asp?Asn?Arg?Glu?Cys?Ile?Ser?Met?Asp?Tyr?Lys
165 170 175
Gln?Thr?Gln?Leu?Cys?Leu?Ile?Gly?Cys?Lys?Pro?Pro?Ile?Gly?Glu?His
180 185 190
Trp?Gly?Lys?Gly?Ser?Pro?Cys?Thr?Asn?Val?Ala?Val?Asn?Pro?Gly?Asp
195 200 205
Cys?Pro?Pro?Leu?Glu?Leu?Ile?Asn?Thr?Val?Ile?Gln?Asp?Gly?Asp?Met
210 215 220
Val?Asp?Thr?Gly?Phe?Gly?Ala?Met?Asp?Phe?Thr?Thr?Leu?Gln?Ala?Asn
225 230 235 240
Lys?Ser?Glu?Val?Pro?Leu?Asp?Ile?Cys?Thr?Ser?Ile?Cys?Lys?Tyr?Pro
245 250 255
Asp?Tyr?Ile?Lys?Met?Val?Ser?Glu?Pro?Tyr?Gly?Asp?Ser?Leu?Phe?Phe
260 265 270
Tyr?Leu?Arg?Arg?Glu?Gln?Met?Phe?Val?Arg?His?Leu?Phe?Asn?Arg?Ala
275 280 285
Gly?Ala?Asp?Trp?Glu?Tyr?Ser?Val?Gly?Glu?Asn?Val?Pro?Asp?Asp?Leu
290 295 300
Tyr?Ile?Lys?Gly?Ser?Gly?Ser?Thr?Ala?Asn?Leu?Ala?Ser?Ser?Asn?Tyr
305 310 315 320
Phe?Pro?Thr?Pro?Ser?Gly?Ser?Met?Val?Thr?Ser?Asp?Ala?Gln?Ile?Phe
325 330 335
Asn?Lys?Pro?Tyr?Trp?Leu?Gln?Arg?Ala?Gln?Gly?His?Asn?Asn?Gly?Ile
340 345 350
Cys?Trp?Gly?Asn?Gln?Leu?Phe?Val?Thr?Val?Val?Asp?Thr?Thr?Arg?Ser
355 360 365
Thr?Asn?Met?Ser?Leu?Cys?Ala?Ala?Ile?Ser?Thr?Ser?Glu?Thr?Thr?Tyr
370 375 380
Lys?Asn?Thr?Asn?Phe?Lys?Glu?Tyr?Leu?Arg?His?Gly?Glu?Glu?Tyr?Asp
385 390 395 400
Leu?Gln?Phe?Ile?Phe?Gln?Leu?Cys?Lys?Ile?Thr?Leu?Thr?Ala?Asp?Val
405 410 415
Met?Thr?Tyr?Ile?His?Ser?Met?Asn?Ser?Thr?Ile?Leu?Glu?Asp?Trp?Asn
420 425 430
Phe?Gly?Leu?Gln?Pro?Pro?Pro?Gly?Gly?Thr?Leu?Glu?Asp?Thr?Tyr?Arg
435 440 445
Phe?Val?Thr?Ser?Gln?Ala?Ile?Ala?Cys?Gln?Arg?His?Thr?Pro?Pro?Ala
450 455 460
Pro?Lys?Glu?Asp?Pro?Leu?Lys?Lys?Tyr?Thr?Phe?Trp?Glu?Val?Asn?Leu
465 470 475 480
Lys?Glu?Lys?Phe?Ser?Ala?Asp?Leu?Asp?Gln?Phe?Pro?Leu?Gly?Arg?Lys
485 490 495
Phe?Leu?Leu?Gln?Ala?Gly?Leu?Lys?Ala?Lys?Pro?Lys?Phe?Thr?Leu?Gly
500 505 510
Lys?Arg?Lys?Ala?Thr?Pro?Thr?Thr?Ser?Ser?Thr?Ser?Thr?Thr?Ala?Lys
515 520 525
Arg?Lys?Lys?Arg?Lys?Leu
530
<210>3
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: outside forward primer F
<400>3
agatctgcca?ccatgtctct?ttggctgcct 30
<210>4
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: outside reverse primer R
<400>4
atgtcgtact accatcacca tcaccatcac 30
<210>5
<211>28
<212>DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: mutagenic primer Fm
<400>5
aagcttttta?
cagcttacg?ttttttgcg 28
<210>6
<211>28
<212>DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: mutagenic primer Rm
<400>6
aactgctaaa
cgcaaaaaac?gtaagctg 28
<210>7
<211>29
<212>DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: upstream primer I
<400>7
gg
ggcgcctc?tctttggctg?cctagtgag 29
<210>8
<211>29
<212>DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: downstream primer I
<400>8
gg
ctcgagtt?acagcttacg?ttttttgcg 29
Claims (8)
1. viral capsid chimeric protein HPV16L1-P
D, have the aminoacid sequence shown in SEQ ID No.2.
2. the described viral capsid chimeric protein of claim 1 HPV16L1-P
DEncoding gene, have the nucleotide sequence shown in SEQ ID No.1.
3. the described viral capsid chimeric protein of claim 1 HPV16L1-P
DThe preparation method, may further comprise the steps:
A.HPV16L1-P
DThe structure of gene
With wild-type HPV16L1 gene is template, adopts overlapping extension PCR method to insert coding P between 798/799 bit base of HPV16L1 gene
DThe base sequence of peptide, design of primers is as follows: outside forward primer F is shown in SEQ ID No.3, and outside reverse primer R is shown in SEQ ID No.4, and mutagenic primer Fm is shown in SEQID No.5, and mutagenic primer Rm is shown in SEQ ID No.6; The PCR product is after agarose gel electrophoresis is identified, the purpose fragment of the about 1600bp of purifying, gained purpose fragment cloning is gone into pCR II-TOPO carrier, be transformed into DH5 α competent escherichia coli cell again, with the LB plate screening positive colony bacterium that contains penbritin, extract plasmid, adopt enzyme to cut and identify positive colony plasmid and order-checking, obtain the HPV16L1-P of 1602bp with the PCR method
DGene has the nucleotide sequence shown in SEQ ID No.1;
B.HPV16L1-P
DThe clone of gene
With step a gained positive colony plasmid is template, PCR method amplification HPV16 L1-P
DGene, design of primers is as follows: upstream primer I is shown in SEQ ID No.7, and downstream primer I is shown in SEQ ID No.8; The PCR product is identified once more according to the described method of step a, purifying, and the clone transforms, and screening is identified, and a series of processes such as order-checking are confirmed clone HPV16 L1-P
DThe order exactness of gene;
C. recombinant vectors pFastBacDual HPV16 L1-P
DStructure
Step b gained positive colony plasmid is carried out double digestion with EheI and XhoI, obtain HPV16 L1-P
DGene fragment is cloned into baculovirus transfer vector pFastBacDual again, obtains recombinant vectors pFastBacDual HPV16 L1-P
D
D. contain HPV16 L1-P
DThe preparation of the recombinant baculovirus of gene
According to BaculoGold
TMTransfection reagent box description operation is with the recombinant vectors pFastBacDual HPV16 L1-P of step c structure
DWith the baculovirus linear DNA be BaculoGold
TMDNA cotransfection insect cell Sf9, homologous recombination obtains to contain HPV16L1-P in born of the same parents
DThe recombinant baculovirus of gene; Transfection Sf9 cell was cultivated 3 days for 27 ℃ in temperature, collected culture supernatant as virus stock solution used;
E. insect cell inner expression and HPV16L1-P
DPurifying
Steps d gained virus stock solution used is infected the Sf9 cell, put temperature and cultivated 3 days for 27 ℃, collect and be subjected to transfect cell; It is among the PBS that the cell precipitation that will be contaminted is resuspended in phosphate buffered saline buffer, ultrasonic broken wall, low-speed centrifugal is collected supernatant, be transferred to and fill in the centrifuge tube that mass percentage concentration is 40% sucrose solution, the ultracentrifugation collecting precipitation, with the CsCl density gradient ultracentrifugation, buoyant density is the white protein band of 1.34g/ml in the collection tube again, promptly gets viral capsid chimeric protein HPV16L1-P
D
4. the described viral capsid chimeric protein of claim 1 HPV16L1-P
DApplication as gene or drug targeting carrier.
5. the described viral capsid chimeric protein of claim 1 HPV16L1-P
DApplication as the vaccine delivery instrument.
6. viral capsid chimeric protein HPV16L1-P according to claim 5
DApplication as the vaccine delivery instrument is characterized in that: described HPV16L1-P
DApplication as systemic lupus erythematous vaccine delivery instrument.
7. the pseudo-viral vaccine of the therapeutic of systemic lupus erythematous, it is characterized in that: described vaccine is with the described viral capsid chimeric protein of claim 1 HPV16L1-P
DDiphtheria toxin A chain under the parcel B cell specificity promotor Ig κ control is a DT-A gene expression profiling plasmid DNA molecule, the pseudo-virus of the simulation natural viral spline structure that self-assembly forms.
8. adopt the described viral capsid chimeric protein of claim 1 HPV16L1-P
DThe method of the pseudo-viral vaccine of the therapeutic of preparation system lupus erythematosus may further comprise the steps:
A. viral capsid chimeric protein HPV16L1-P
DPreparation
Be prepared according to the described method of claim 3;
B. the preparation of recombinant vectors pcDNA3 Ig κ DT-A
The pTHA45 carrier contains Ig κ DT-A gene fragment, with Apa I and Bgl II double digestion pTHA45 carrier, cut glue and reclaim purifying Ig κ DT-A gene fragment, be connected with the pcDNA3 carrier that digests through same double digestion again, obtain recombinant vectors pcDNA 3I g κ DT-A;
C. the preparation of the pseudo-viral vaccine of the therapeutic of systemic lupus erythematous
Get step a gained viral capsid chimeric protein HPV16L1-P
D, add denaturing agent and make protein denaturation, add step b gained recombinant vectors pcDNA3Ig κ DT-A again, dialysed overnight in PBS makes metaprotein renaturation gradually, HPV16L1-P in renaturation process
DParcel recombinant vectors pcDNA3Ig κ DT-A, self-assembly forms the pseudo-viral vaccine of simulation natural viral spline structure.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA200810069886XA CN101314615A (en) | 2008-06-25 | 2008-06-25 | Viral capsid mosaic protein HPV16 L1-P<D>, preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA200810069886XA CN101314615A (en) | 2008-06-25 | 2008-06-25 | Viral capsid mosaic protein HPV16 L1-P<D>, preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107119058A (en) * | 2017-05-10 | 2017-09-01 | 斯澳生物科技（苏州）有限公司 | A kind of MS2 capsid proteins fusion expression method and its application |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107119058A (en) * | 2017-05-10 | 2017-09-01 | 斯澳生物科技（苏州）有限公司 | A kind of MS2 capsid proteins fusion expression method and its application |
| CN107119058B (en) * | 2017-05-10 | 2020-12-08 | 斯澳生物科技（苏州）有限公司 | MS2 capsid protein fusion expression method and application thereof |
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Application publication date: 20081203 |