CN101475640B - Conjugate, and preparation and use thereof - Google Patents
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- CN101475640B CN101475640B CN2009100770585A CN200910077058A CN101475640B CN 101475640 B CN101475640 B CN 101475640B CN 2009100770585 A CN2009100770585 A CN 2009100770585A CN 200910077058 A CN200910077058 A CN 200910077058A CN 101475640 B CN101475640 B CN 101475640B
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Abstract
The invention discloses a coupling object. The coupling object is obtained through the coupling of at least one protein in the four proteins of a recombined human epidermal growth factor and a recombined epidermal growth factor mutant with a carrier protein. The amino acid sequence of the recombined human epidermal growth factor is shown by the No.2 sequence in a sequence list. The recombined epidermal growth factor mutant can be one of the following proteins: 1)the protein obtained through the deletion of the No.53 arginine in the No.2 sequence in the sequence list; 2) the protein obtained through the deletion of the No.53 arginine and No.52 leucine in the No.2 sequence in the sequence list; and 3) the protein obtained through the addition of methionine before the No.1 asparagine in the No.2 sequence in the sequence list. The coupling object can be used as a vaccine for the active treatment of an epithelium sourced tumor after being mixed with an adjuvant. Results show that the vaccine can induct the high level antibodies in a mouse; the highest antibody titer reaches 1:20,000; and the prolonging rate of the survival period of the mouse reaches 69.4 percent.
Description
Technical field
The present invention relates to a kind of conjugate and preparation method thereof and application.
Background technology
According to the report of the World Health Organization, the annual de novo tumour patient in the whole world has 1,000 ten thousand, dead 6,000,000 approximately at present.Since the seventies in 20th century, China's malignant tumour M ﹠ M is in rising trend always, and the pathogenesis of cancer number about 2,000,000, dead 1,500,000 in 2000.The tumor mortality rate surpasses cardiovascular and cerebrovascular disease and leaps to the first at present.Malignant tumour has become human " first killer ".
Studies show that, epithelium source property tumour, comprise malignant tumours such as lung cancer, cancer of the stomach, liver cancer, the esophageal carcinoma, colorectal cancer, cervical cancer, nasopharyngeal carcinoma and mammary cancer, its generation, formation, propagation, transfer and prognosis mala etc. are all relevant with GG hEGF/EGF-R ELISA (EGF/EGFR) signalling channel.At epithelium source property tumor cell surface, therefore the EGFR overexpression, is compared with normal cell, and the EGF/EGFR signalling channel of epithelium source property tumour cell too activates, and causes the undying propagation of tumour cell, differentiation, and finally forms noumenal tumour.Therefore, be target spot with the EGF/EGFR signalling channel, reduce the activity of this signalling channel, suppress propagation, the differentiation of tumour cell, finally reach epithelium source property tumour is carried out specific treatment, become the focus of this area research.At present, be that the epithelium source property tumour-specific treatment means of target spot mainly comprises the studies on Monoclonal Antibody of anti-EGFR and to the research of tyrosine kinase inhibitor in the signalling channel at the EGF/EGFR signalling channel.
Monoclonal antibody worldwide is widely used as a kind of main means of the passive treatment of cancer.In the property tumour of epithelium source, tumor cell surface EGFR overexpression, therefore, the monoclonal antibody of anti-EGFR and the intravital EGF of people are competitively with after EGFR combines, the activity that suppresses the EGF/EGFR signalling channel, thereby propagation, the differentiation of inhibition tumour cell, and then the purpose of arrival cancer therapy.At present, the whole world three kinds of monoclonal antibodies at EGFR of having gone on the market are used for the epithelium source property tumor treatment of EGFR high expression level, have obtained obvious effects.Three kinds of monoclonal antibody and indications thereof at EGFR are as shown in the table:
But owing to monoclonal antibody costs an arm and a leg, and need multiple injection, comprise that therefore there is certain difficulty in developing countries' general patient use of China.
Studies show that, EGF with can activating cells after EGFR combines in tyrosine kinase activity, thereby make tumor cell proliferation, differentiation.Therefore, suppressing the intracellular tyrosine kinase activity also is a kind of approach of epithelium source property oncotherapy.Te Luokai (Erlotinib) and Iressa (Gefitinib) are two kinds of oral EGF-R ELISA-Tyrosylprotein kinases (EGFR-TK) antagonists, the two wires or the three-way treatment plan of the advanced NSCLC that the former failed to respond to any medical treatment as standard scheme through drugs approved by FDA in September, 2002, the latter is used for the treatment of nonsmall-cell lung cancer in late period as three-way single medicine through drugs approved by FDA in May, 2003.But these two kinds of small-molecule drugs easily produce resistance in therapeutic process, and there is dispute in the patient treatment effect of different ethnic groups, different living environments.
Owing to have various deficiencies at monoclonal antibody, the small molecules tyrosine kinase inhibitor of EGF/EGFR signalling channel, therefore, the medicine of developing a kind of evident in efficacy, the cheap epithelium source that treats and/or prevents property tumour becomes active demand.
In the EGF/EGFR system, the activation of signalling channel is EGF and finish jointly after the EGFR specificity combines.Because monoclonal antibody against EGFR can reach inhibition signalling channel activity by emulative the combination with EGFR, the antibody of pointing out anti-EGF is with after the EGF specificity combines, also may play and suppress the activation of EGF/EGFR signalling channel, the differentiation of inhibition tumor cell proliferation, finally reach the purpose of oncotherapy.
Recombinant human epidermal growth factor is one of cytokine of human body self generation, has the various biological function.Because immunological tolerance, human body can not produce immunne response to the free Urogastron.Along with immunology research and vaccine development Progress in technique, it is found that weak immunogenic polysaccharide antigen (as pneumonia polysaccharide, the scorching polysaccharide of C group's neisseria meningitis, hemophilus influenzae polysaccharide) with the suitable carriers albumen coupling after, can increase immunogenicity of antigens, make body produce the memory immunne response such antigen.Research shows simultaneously, behind micromolecular polypeptide and the carrier protein couplet, can increase the immunogenicity of polypeptide equally.
In Chinese patent 95115090.1, the researchist forms conjugate with Urogastron (EGF) by chemical process and Toxoid,tetanus coupling, and experimentation on animals shows, with above-mentioned conjugate immune mouse, produces high-caliber anti-EGF antibody.In Chinese patent 03818573.3 (WO03/106487A1), the researchist will be from the polypeptide fragments and the coupling of keyhole worm relative hemocyanin of vascular endothelial growth factor (VEGF), experimentation on animals shows, with above-mentioned conjugate immune mouse, not only produce the antibody of high-caliber anti-VEGF polypeptide fragment, and can significantly suppress the ability that tumour cell shifts.
Summary of the invention
The purpose of this invention is to provide a kind of conjugate and preparation method thereof and application.
Conjugate provided by the present invention is that at least a and carrier protein couplet in recombinant human epidermal growth factor and these four kinds of albumen of following three kinds of recombinant human epidermal growth factor mutant is obtained;
The aminoacid sequence of described recombinant human epidermal growth factor is shown in sequence in the sequence table 2;
Described recombinant human epidermal growth factor mutant is following any albumen:
1) the 53rd of sequence 2 the albumen that the arginine disappearance obtains in the sequence table;
2) the 53rd of sequence 2 the arginine and the 52nd albumen that the leucine disappearance obtains in the sequence table;
3) add the albumen that methionine(Met) obtains before the 1st of sequence 2 the l-asparagine in the sequence table.
In the above-mentioned conjugate, described carrier proteins is Neisseria meningitidis outer membrane protein, diphtheria toxoid, Toxoid,tetanus or heat shock protein(HSP);
The structural formula of described conjugate is:
Xm(rhEGF)n；
Wherein, X is a carrier proteins, and rhEGF is that recombinant human epidermal growth factor and these four kinds of described three kinds of recombinant human epidermal growth factor mutant are proteic at least a;
M is the integer of 1-5, and n is the integer of 1-12; Preferred m is the integer of 1-4, and n is the integer of 1-8; Further preferred m is the integer of 1-3, and n is the integer of 1-6.
In the above-mentioned conjugate, described carrier proteins specifically can be the Neisseria meningitidis outer membrane protein, and the aminoacid sequence of described Neisseria meningitidis outer membrane protein is shown in sequence in the sequence table 4.
The structural formula of above-mentioned conjugate specifically can be:
(P64k)
2(rhEGF)
5；
Wherein, P64K is the Neisseria meningitidis outer membrane protein, and rhEGF is the mixture of described recombinant human epidermal growth factor mutant;
The mixture of described recombinant human epidermal growth factor mutant is made up of following two kinds of albumen:
1) the 53rd of sequence 2 the albumen that the arginine disappearance obtains in the sequence table;
2) the 53rd of sequence 2 the arginine and the 52nd albumen that the leucine disappearance obtains in the sequence table.
The mixture of described recombinant human epidermal growth factor mutant changes the recombinant human epidermal growth factor encoding gene to express in the yeast over to and obtains;
The nucleotide sequence of described recombinant human epidermal growth factor encoding gene is shown in sequence in the sequence table 1;
Described yeast specifically can be pichia spp X33.
Another object of the present invention provides the preparation method of above-mentioned conjugate.
The preparation method of conjugate provided by the present invention, be that at least a in described recombinant human epidermal growth factor and these four kinds of albumen of described three kinds of recombinant human epidermal growth factor mutant mixed with carrier proteins, add coupling agent in mixture, reaction obtains conjugate.
In the aforesaid method, the mol ratio of described recombinant human epidermal growth factor and described three kinds of recombinant human epidermal growth factor mutant and described carrier proteins is (1-20): 1, be preferably (5-15): and 1, more preferably (8-12): 1;
Described coupling agent can be congenerous coupling agent or bifunctional coupling agent, is preferably the congenerous coupling agent, specifically can be glutaraldehyde.
The ultimate density of described glutaraldehyde in mixture is 0.01-0.1%, is preferably 0.02-0.08%, more preferably 0.03-0.06%;
The described link coupled reaction times is 20-90 minute, is preferably 30-60 minute.
Above-mentioned coupling process can be finished in liquid phase, also can finish at solid phase surface.During the solid phase coupling, can be earlier the mixture or the P64k of described recombinant human epidermal growth factor mutant be bonded to dielectric surface, after the glutaraldehyde activation, again with another protein binding to dielectric surface, make two kinds of albumen finish coupling process.
Aforesaid method comprises that also the conjugate that will obtain carries out purifying, with the step of the mixture of removing glutaraldehyde and described recombinant human epidermal growth factor mutant.In the conjugate behind the purifying, the quality percentage composition of conjugate can reach 50%, is preferably 70%, and more preferably 75%; Wherein, the quality percentage composition of the mixture of free recombinant human epidermal growth factor mutant is not more than 5%, is preferably and is not more than 1%.Purification process can adopt current purification method, is preferably ultrafiltration purification, and the molecular weight cut-off of ultra-filtration membrane bag is 50kDa.
The 3rd purpose of the present invention provides a kind of vaccine that treats and/or prevents epithelium source property tumour.
The vaccine that treats and/or prevents epithelium source property tumour provided by the present invention, its activeconstituents are above-mentioned any conjugate.
The present invention is prepared into conjugate by mixture (rhEGF) and Neisseria meningitidis outer membrane protein (P64k) coupling with the recombinant human epidermal growth factor mutant.This conjugate mixes the back is used for epithelium source property tumour as vaccine active treatment with immunological adjuvant, make the patient produce specific antibody at EGF, with EGF content, downward modulation EGF/EGFR signalling channel activity in reaching and among the patients serum, suppress epithelium source property tumor cell proliferation, differentiation, finally reach the purpose of epithelium source property tumour active immunity treatment.
In the above-mentioned vaccine, described adjuvant is aluminium adjuvant or Momtanide ISA 51 adjuvants, is preferably Momtanide ISA 51 adjuvants.Momtanide ISA 51 is a kind of novel human immunological adjuvants, form by the height purified glyceryl alcohol acid anhydride oleic acid ester (Montanide 80) that is dissolved in mineral oil solution (Drakeol 6VR), can form water in oil emulsion with antigen emulsification, play slow releasing function, and enhancement antigen is the former picked-up of delivery cell antagonism, the immunogenicity of enhancement antigen.
Described epithelium source property tumour comprises nonsmall-cell lung cancer, cancer of the stomach, liver cancer, the esophageal carcinoma, colorectal cancer or nasopharyngeal carcinoma.
It is action target spot with the cell signal passage that vaccine of the present invention utilizes the method for specificity active immunity, produce anti-EGF antibody, corresponding immunity system is stimulated, recover patient's self immunity system, thereby suppress effectively, block and regulated the EGF/EGFR system, the epithelium source property tumour of EGFR overexpression is had good result of treatment.Compare with traditional simple chemicotherapy method, the toxic side effect of vaccine of the present invention is very little, safe, better tolerance, and can give full play to cancer patients's self antitumor potential, reaches the purpose of oncotherapy.Use vaccine immune mouse of the present invention, high-level antibody in the energy inducing mouse body, the highest antibody titers reaches 1: 20000, and the survival time of mouse prolongation ratio reaches 69.4%.
Description of drawings
Fig. 1 is the agarose gel electrophoresis result of recombinant human epidermal growth factor pcr amplification product
Fig. 2 is the agarose gel electrophoresis result of recombinant human epidermal growth factor double digestion product
Fig. 3 is the reverse-phase chromatography collection of illustrative plates of recombinant human epidermal growth factor mutant mixture
Fig. 4 is the peptide spectrum (trypsinase) of recombinant human epidermal growth factor mutant mixture
Fig. 5 is the electrophoretogram of recombinant human epidermal growth factor mutant mixture
Fig. 6 is the mass spectroscopy collection of illustrative plates of recombinant human epidermal growth factor mutant mixture
Fig. 7 is the agarose gel electrophoresis result of Neisseria meningitidis outer membrane protein P64k pcr amplification product
Fig. 8 is the reverse-phase chromatography collection of illustrative plates of Neisseria meningitidis outer membrane protein P64k
Fig. 9 is the peptide spectrum (trypsinase) of Neisseria meningitidis outer membrane protein P64k
Figure 10 is the electrophoretogram of Neisseria meningitidis outer membrane protein P64k
Figure 11 is the mass spectroscopy collection of illustrative plates of Neisseria meningitidis outer membrane protein P64k
Figure 12 is the gel chromatography collection of illustrative plates of conjugate rhEGF-P64k
Figure 13 is that collection of illustrative plates is differentiated in the immunity of each component among the conjugate rhEGF-P64k
Figure 14 is the molecular weight detection collection of illustrative plates of conjugate rhEGF-P64k
Figure 15 is conjugate (P64K)
2(rhEGF)
5With the electronic microscope photos result after the adjuvant Montanide ISA51 emulsification
Figure 16 is conjugate (P64K)
2(rhEGF)
5Immunogenicity analysis in non-human primate
Embodiment
Experimental technique described in the following embodiment if no special instructions, is ordinary method; Described reagent and biomaterial if no special instructions, all can obtain from commercial channels.
The preparation of embodiment 1, recombinant human epidermal growth factor mutant mixture (rhEGF)
1, the clone of recombinant human epidermal growth factor encoding gene
According to zymic codon bias, the full length DNA sequence of synthetic encoding mature recombinant human epidermal growth factor, with this unnamed gene be its nucleotide sequence shown in sequence in the sequence table 1, its amino acid sequence coded is shown in sequence in the sequence table 2.
Full length DNA sequence with above-mentioned synthetic recombinant human epidermal growth factor is a template, uses Primer 5.0 software design primers: upstream primer: 5-CCG
CTCGAGAACTCAGATAGTGAATGCC-3, downstream primer: 5-CCG
GAATTCTCAACGTAATTCCCACCA-3, wherein underscore partly is respectively the restriction enzyme site of XhoI and EcoRI, carries out pcr amplification.The PCR reaction system is: 0.5 μ l template, 10 * PCR damping fluid, 5 μ l, 10mmol/LdNTP 1 μ l, Pyrobest high-fidelity DNA polymerase (chemical product is given birth in Shanghai) 1 μ l, final concentration is each 0.5 μ l of upstream and downstream primer of 0.5 μ mol/L, adds ultrapure water to 50 μ l.The PCR reaction conditions is: 94 ℃ of pre-sex change 5min of elder generation; 94 ℃ of sex change 30s then, 56 ℃ of annealing 30s, 72 ℃ are extended 30s; Totally 30 circulations, last 72 ℃ are extended 10min.Pcr amplification product is carried out 1% agarose gel electrophoresis, and the result as shown in Figure 1.Wherein, swimming lane 1 is the pcr amplification product of recombinant human epidermal growth factor encoding gene, and swimming lane 2 is the DL2000DNA molecular weight standard.The result shows that amplification obtains the recombinant human epidermal growth factor encoding gene about 160bp.
2, the structure of carrier and engineering strain
Reclaim the above-mentioned purpose fragment, the PCR product and the pPICZa carrier (available from Invitrogen company) that reclaim are carried out double digestion with XhoI and EcoRI respectively, the enzyme of the PCR product of above-mentioned steps 1 behind XhoI and EcoRI double digestion cut product and carried out the detection of 1% agarose gel electrophoresis, and the result as shown in Figure 2.Wherein, swimming lane 1 is the DL2000DNA molecular weight standard, and swimming lane 2 is cut product for the enzyme of PCR product behind XhoI and EcoRI double digestion of above-mentioned steps 1.Cut glue and reclaim endonuclease bamhi, the enzyme that enzyme is cut product and carrier pPICZa is cut product with 1: 10 mixed in molar ratio, connects through T4DNA ligase enzyme room temperature and spends the night connection product CaCl
2Method transforms the TOP10F competent cell, coats on the LB flat board that contains Zeocin (25 μ g/ml), and the picking mono-clonal, XhoI and EcoRI double digestion are identified positive colony.
Extract the DNA of above-mentioned positive colony, linearizing after Bgl II enzyme is cut, the electricity consumption method for transformation changes in the competent cell of pichia spp X33 (available from Invitrogen company), the final concentration of coating Zeocin is respectively the dull and stereotyped enterprising row filter of YPDS of 100 μ g/mL, 500 μ g/mL and 1000 μ g/mL, be inverted for 28～30 ℃ and cultivate 3～10d, until there being mono-clonal to grow, obtain express recombinant human epidermal growth factor's Yeast engineering bacteria.
3, the purifying of engineering strain large scale culturing, recombinant human epidermal growth factor and detection
The express recombinant human epidermal growth factor's that above-mentioned steps 2 is obtained Yeast engineering bacteria is cultivated through three grades and is amplified, and goes at last in the fermentor tank of 1000L, and the fermentor tank parameter is set to: stirring velocity 350-400rpm, temperature 29-32 ℃, dissolved oxygen is controlled at 30-40%.Cultivate after 72-96 hour, stop cultivating.
With nutrient solution through the centrifugal solid-liquid separation of carrying out of continuous high speed.Collect supernatant liquor and carry out anion-exchange chromatography (DEAE-Sepharose FF GE), hydrophobic chromatography (Phenyl Sepharose CL-4B) and cation-exchange chromatography (SP-Sepharose FF GE) successively, obtain recombinant human epidermal growth factor stoste.
According to 2005 editions the 3rd requirements of the Pharmacopoeia of the People's Republic of China and quality standard the recombinant human epidermal growth factor stoste of above-mentioned acquisition is carried out purity, residual impurity and structure and differentiate, concrete detected result such as Fig. 3-shown in Figure 6.The result shows, containing the recombinant human epidermal growth factor C-terminal in the above-mentioned recombinant human epidermal growth factor stoste that obtains and lack arginic mutant and recombinant human epidermal growth factor C-terminal disappearance arginine and leucic mutant, is the mixture of these two kinds of recombinant human epidermal growth factor mutant.Wherein, Fig. 3 is the reverse-phase chromatography collection of illustrative plates of recombinant human epidermal growth factor stoste, the detection wavelength is 214nm, peak 2 lacks arginic mutant for the recombinant human epidermal growth factor C-terminal, its chromatographic retention is 26.04 minutes, peak 1 is recombinant human epidermal growth factor C-terminal disappearance arginine and leucic mutant, and its chromatographic retention is 22.06 minutes, and the quality percentage composition at peak 1 and peak 2 reaches 97.9%; Fig. 4 is the peptide spectrum (trypsinase) of recombinant human epidermal growth factor mutant mixture, the result shows that the collection of illustrative plates of the recombinant human epidermal growth factor mutant mixture of above-mentioned acquisition and recombinant human epidermal growth factor standard substance (Cuba molecular immunology center) is in full accord; Fig. 5 is the electrophoretogram of recombinant human epidermal growth factor mutant mixture, EGF represents the recombinant human epidermal growth factor mutant mixture that the Yeast engineering bacteria of above-mentioned acquisition is expressed, LMWP represents low molecular weight protein (LMWP) standard substance (available from GE company), in electrophoretogram, the recombinant human epidermal growth factor mutant mixture of above-mentioned acquisition has only a band, and purity is 100%; Fig. 6 is the mass spectroscopy collection of illustrative plates of recombinant human epidermal growth factor mutant mixture, molecular weight is that the component of 6060.7Da is that C-terminal lacks arginic recombinant human epidermal growth factor mutant, and molecular weight is that the component of 5947.4Da is C-terminal disappearance arginine and leucic recombinant human epidermal growth factor mutant.
The preparation of embodiment 2, Neisseria meningitidis outer membrane protein (P64k)
1, the clone of Neisseria meningitidis outer membrane protein encoding gene
According to the sequence of the Neisseria meningitidis of GenBank Accession No.X77920.1, adopt Primer 5.0 software design primers: upstream primer: 5-CATG
CCATGGCTTTAGTTGAATTGAA-3, downstream primer: 5-CCG
GAATTCTTATTTTTTCTTTTGCGGAG-3, wherein underscore partly is respectively the restriction enzyme site of NcoI and EcoRI.Neisseria meningitidis CMCC 29336 (available from Chinese medicine bacterium preservation center, being called for short CMCC) is boiled 10min under 100 ℃ of conditions, draw 3 μ l, carry out pcr amplification as template.The PCR reaction system is: 3 μ l templates, 10 * PCR damping fluid, 5 μ l, 10mmol/L dNTP 1 μ l, Pyrobest high-fidelity DNA polymerase (chemical product is given birth in Shanghai) 1 μ l, final concentration is each 0.5 μ l of upstream and downstream primer of 0.5 μ mol/L, adds ultrapure water to 50 μ l.The PCR reaction conditions is: 94 ℃ of pre-sex change 5min of elder generation; 94 ℃ of sex change 45s then, 50 ℃ of annealing 45s, 72 ℃ are extended 2min; Totally 30 circulations, last 72 ℃ are extended 10min.Pcr amplification product is carried out 1% agarose gel electrophoresis, and the result as shown in Figure 7.Wherein, swimming lane 1 is the DL2000DNA molecular weight standard, and swimming lane 2 is the pcr amplification product of Neisseria meningitidis outer membrane protein (P64k) encoding gene.The result shows that amplification obtains the encoding gene of the Neisseria meningitidis outer membrane protein about 1800bp.Encoding gene to the Neisseria meningitidis outer membrane protein of above-mentioned acquisition checks order, and sequencing result shows that its nucleotide sequence is shown in sequence in the sequence table 3, and its amino acid sequence coded is shown in sequence in the sequence table 4.
2, the structure of carrier and engineering strain
Reclaim the above-mentioned purpose fragment, the PCR product and the pET28a carrier that reclaim are carried out double digestion with NcoI and EcoRI respectively, the enzyme of the PCR product of above-mentioned steps 1 behind NcoI and EcoRI double digestion cut product and carried out the detection of 1% agarose gel electrophoresis, cut glue and reclaim endonuclease bamhi, the enzyme that enzyme is cut product and carrier pET28a is cut product with 1: 3 mixed in molar ratio, spend the night through the connection of T4DNA ligase enzyme room temperature, connect product CaCl
2Method transforms the JM109 competent cell, after 12 hours, selects mono-clonal, with bacterium colony PCR method screening positive clone, obtains expressing the colibacillus engineering of Neisseria meningitidis outer membrane protein (P64k).
3, purifying and the detection of engineering strain large scale culturing, Neisseria meningitidis outer membrane protein P64k
The colibacillus engineering of the expression Neisseria meningitidis outer membrane protein P64k that above-mentioned steps 2 is obtained is cultivated through three grades and is amplified, go at last in the broth culture of 500L, the fermentor tank parameter is set to: stirring velocity 350-400rpm, temperature 35-37 ℃, dissolved oxygen is controlled at 30-40%.Cultivate after 36-48 hour, stop cultivating.
With nutrient solution through the centrifugal solid-liquid separation of carrying out of continuous high speed to collect thalline, with the thalline collected behind high-pressure homogenization, centrifugal removal bacterial chip, in supernatant liquor, add solid ammonium sulfate, carry out hydrophobic chromatography, anion-exchange chromatography (Q-Sepharose FF GE) and gel-filtration (Supperdex S200 GE) successively, obtain Neisseria meningitidis outer membrane protein stoste.
According to 2005 editions the 3rd requirements of the Pharmacopoeia of the People's Republic of China and quality standard the Neisseria meningitidis outer membrane protein stoste of above-mentioned acquisition is carried out purity, residual impurity and structure and differentiate, concrete detected result such as Fig. 8-shown in Figure 11.Wherein, Fig. 8 is the reverse-phase chromatography collection of illustrative plates of Neisseria meningitidis outer membrane protein (P64k), and the detection wavelength is 280nm, the result shows, the Neisseria meningitidis outer membrane protein of above-mentioned acquisition has only a peak, and its chromatographic retention is 7.71 minutes, and chromatographic purity is 100%; Fig. 9 is the peptide spectrum (trypsinase) of Neisseria meningitidis outer membrane protein P64k, and the result shows that the Neisseria meningitidis adventitia egg P64k of above-mentioned acquisition collection of illustrative plates white and P64k standard substance (Cuba molecular immunology center) are in full accord; Figure 10 is the electrophoretogram of Neisseria meningitidis outer membrane protein P64k, P64K represents the Neisseria meningitidis outer membrane protein P64k that the colibacillus engineering of above-mentioned acquisition is expressed, LMWP represents low molecular weight protein (LMWP) standard substance (available from GE company), in electrophoretogram, the Neisseria meningitidis outer membrane protein P64k of above-mentioned acquisition has only a band, and purity is 100%; Figure 11 is the mass spectroscopy collection of illustrative plates of Neisseria meningitidis outer membrane protein P64k, and the mass spectrum molecular weight is 61932.7Da, and is consistent with the theoretical molecular that aminoacid sequence is calculated.
The preparation of embodiment 3, recombinant human epidermal growth factor mutant mixture and Neisseria meningitidis outer membrane protein conjugate (rhEGF-P64k)
With the dobell's solution (pH8.5) of 0.1M the concentration of the P64k of the mixture of the recombinant human epidermal growth factor mutant of the foregoing description 1 preparation and the foregoing description 2 preparations is adjusted to 1.0mg/mL respectively, the concentration of getting different volumes be the mixture of recombinant human epidermal growth factor mutant of 1.0mg/mL and P64k protein solution to the aseptic pyrogen-free reactor of 10L, make the mixture of recombinant human epidermal growth factor mutant and the mol ratio of P64k be (12-8): 1.Above-mentioned mixed solution mixed in reactor and balance to room temperature, slowly drip volumn concentration and be 0.5% glutaraldehyde solution, make that the final concentration of glutaraldehyde is respectively 0.01%, 0.05% and 0.1% in the mixed solution.After stirring at room 30-60 minute, add the glycine of 0.2M, making its final concentration in the induction system is 5% to stop linked reaction.Obtain the conjugate rhEGF-P64k of recombinant human epidermal growth factor mutant mixture and Neisseria meningitidis outer membrane protein.Conjugate rhEGF-P64k is the phosphate buffered saline buffer that 10 volumes of liquid are changed in the ultra-filtration membrane bag equal-volume ultrafiltration of 50kDa through molecular weight cut-off, and adjusts conjugate concentration to 1.0mg/mL, and 0.2 μ m filter membrane goes out bacterium and filters, and can is preserved.
The structural analysis of the conjugate (rhEGF-P64k) of embodiment 4, recombinant human epidermal growth factor mutant mixture and Neisseria meningitidis outer membrane protein
1, the discriminating of active principle and content analysis
Adopt gel chromatography to identify each components contents in the conjugate of the foregoing description 3 preparations.Chromatographic condition is: chromatographic column: TSK2000SW
XLMoving phase: PBS and quality percentage composition are 0.1% SDS, pH6.7; Detect wavelength: 214nm.
Because Neisseria meningitidis outer membrane protein (P64k) has stronger hydrophobicity, in physiological buffer solution, easily form oligomer, thereby disturb separation and judgement each component of conjugate.Therefore, be 0.1% SDS by in moving phase, adding the quality percentage composition, to eliminate the hydrophobic interaction between the P64k albumen, each component reaches baseline separation in the conjugate thereby make.The gel chromatography collection of illustrative plates of conjugate rhEGF-P64k as shown in figure 12.
The result shows, two peaks appear in the gel chromatography collection of illustrative plates of conjugate rhEGF-P64k, the retention time of main peak is respectively 5.675min (Peak 1) and 7.044min (Peak 2), wherein retention time is the component of 5.675min (Peak 1), apparent molecular weight is conjugate rhEGF-P64k greater than P64k; Retention time is the component of 7.044min (Peak 2), and apparent molecular weight is between P64k and rhEGF, and deduction is the polymkeric substance of recombinant human epidermal growth factor mutant mixture.
In order to verify above deduction, two peaks (5.675min and 7.044min) of collecting conjugate rhEGF-P64k respectively carry out the immune telling test of each component, and the result as shown in figure 13.Wherein, the immune identification result at the peak of collecting among Peak 1 expression Figure 12 1, the immune identification result at the peak of collecting among Peak 2 expression Figure 12 2, conjugate represent gel chromatography separate before the immune identification result of peak 1 and peak 2 mixtures.
The result shows that the antibody of 5.675min component antagonism recombinant human epidermal growth factor and anti-P64k all is positive, and shows that it is conjugate rhEGF-P64k; And the 7.044min component only resists the antibody of recombinant human epidermal growth factor and is positive, and according to its chromatogram relative retention time, then this component is the polymer of recombinant human epidermal growth factor mutant mixture.
According to the chromatographic separation and the immune identification result of conjugate, in the conjugate behind the purifying, the content of active principle rEGF-P64k reaches as high as 75%.
2, conjugate structural characterization
Because conjugate rhEGF-P64K is formed by recombinant human epidermal growth factor mutant mixture and P64K coupling, therefore, as long as accurately determine the molecular weight of conjugate rhEGF-P64K, just can extrapolate recombinant human epidermal growth factor mutant and the shared quality of P64K in the conjugate according to the molecular weight of recombinant human epidermal growth factor mutant and P64K, thereby infer the mol ratio of recombinant human epidermal growth factor mutant and P64K in the conjugate, promptly conjugate rhEGF-P64K is made of several recombinant human epidermal growth factor mutant and several P64K albumen.
Adopt gel chromatography and multi-angle laser light scattering instrument (Multi-Anlge Laser Scattering) polyphone to measure the molecular weight of conjugate rhEGF-P64K.In analytic process, sample is after the gel chromatography fractional separation, successively by laser light scattering instrument (DAWN EOS, Wyatt) and differential detector (OPTILAB DSP, Wyatt), the molecular weight that analysis software (Astra) goes out conjugate rhEGF-P64K according to the laser signal of gathering and differential calculated signals, the molecular weight detection collection of illustrative plates of conjugate rhEGF-P64k as shown in figure 14.
The result shows, the P64k that the multiple angle laser light scattering method is measured and the molecular weight of recombinant human epidermal growth factor mutant are respectively 62.2kDa and 6.3kDa, with the molecular weight of the mass spectroscopy (P64k:61.9kDa that matches; RhEGF:6.0kDa); The molecular-weight average of the conjugate rhEGF-P64K that the multiple angle laser light scattering method is measured is 156.1kDa.
In conjugate rhEGF-P64K, if the mole number of P64k is 1 (m=1), then the quality of recombinant human epidermal growth factor mutant is 93.9kDa (a rhEGF quality=conjugate quality-P64k quality), its mole number is 14.9, i.e. P64k protein molecular surface bonding 14.9 molecular recombination human epidermal growth factor mutant, and the mol ratio of recombinant human epidermal growth factor mutant and P64k is (12-8) when linked reaction: 1, and therefore this possibility does not exist; If the mole number of P64k is 3 (m=3), then the shared quality of P64k reaches 186.6kDa in conjugate, and the conjugate molecular weight has only 156.1kDa, so this may also the existence; If the mole number of P64k is 2 (m=2), the quality of recombinant human epidermal growth factor mutant mixture is 31.7kDa, and its mole number is 5.03, i.e. P64k protein molecular surface bonding 2.5 molecular recombination human epidermal growth factor mutant.This result and process I
125The conjugate analytical results of r-EGF mark is consistent.
The multiple angle laser light scattering analytical results shows, the molecular-weight average of conjugate rhEGF-P64K is 156.1kDa, by two P64k protein moleculars and 5 polymkeric substance that the recombinant human epidermal growth factor mutant molecule is formed, 2.5 recombinant human epidermal growth factor mutant molecules of an average P64k protein molecular surface bonding.The structural formula of conjugate can be expressed as:
(P64k)
2(rhEGF)
5。
1, conjugate (P64K)
2(rhEGF)
5Mix with adjuvant
(1) conjugate (P64K)
2(rhEGF)
5Mix with Montanide ISA 51, emulsification
Get 1.0mL oil adjuvant Montanide ISA 51 with disposable sterilized pyrogen-free syringe, respectively with the conjugate (P64K) of the foregoing description 3 preparation of isopyknic 1.0mg/mL
2(rhEGF)
5Or the recombinant human epidermal growth factor mutant mixture of isopyknic the foregoing description 1 preparation is mixed in the sample bottle, blow and beat repeatedly 20 times, make it to form water in oil mixture, the mixture after blowing and beating repeatedly is added drop-wise in the distilled water, the emulsification drop that is white in color, and do not disperse.Conjugate (P64K)
2(rhEGF)
5With electronic microscope photos result after the adjuvant Montanide ISA51 emulsification as shown in figure 15.The result shows, conjugate (P64K)
2(rhEGF)
5With adjuvant Montanide ISA51 fully emulsified after, be the bead of homogeneous.
(2) conjugate (P64K)
2(rhEGF)
5With the mixing of aluminum hydroxide adjuvant, absorption
Get the conjugate (P64K) of the foregoing description 3 preparations of 1.0mg/mL respectively
2(rhEGF)
5Or the recombinant human epidermal growth factor mutant mixture (being diluted to 1.0mg/mL) of isopyknic the foregoing description 1 preparation is in aseptic pyrogen-free container, adding isopyknic aluminum ion quality percentage composition is the aluminum hydroxide adjuvant of 2.0mg/mL, stirring at room 1 hour.
2, immune programme for children
Get 70 10 age in week BALB/C mice be divided into seven groups at random, be respectively the conjugate group of adjuvant Montanide ISA51 emulsive conjugate group, adjuvant Montanide ISA51 emulsive recombinant human epidermal growth factor mutant mixture group, aluminum hydroxide adjuvant absorption, recombinant human epidermal growth factor mutant mixture group, conjugate group, recombinant human epidermal growth factor mutant mixture group and the negative control group (PBS) of aluminum hydroxide adjuvant absorption.Get the adjuvant Montanide ISA51 and the conjugate (P64K) of 1 preparation of 0.2mL above-mentioned steps respectively
2(rhEGF)
5Mixture, aluminum hydroxide adjuvant and the conjugate (P64K) of mixture, adjuvant Montanide ISA51 and recombinant human epidermal growth factor mutant mixture
2(rhEGF)
5Mixture, the conjugate (P64K) of mixture, aluminum hydroxide adjuvant and recombinant human epidermal growth factor mutant mixture
2(rhEGF)
5, rhEGF and PBS subcutaneous injection immune balb/c mice, immunity in per 7 days once, immunity is 3 times altogether.Last immunity sample of blood, drawn after 7 days, separation of serum is used for antibody titers and detects.
3, antibody titers detects
Adopt the ELISA method to measure the titre of the anti-EGF antibody of specificity in the serum of above-mentioned steps 2 acquisitions.Porous plate wraps quilt with EGF earlier, and the serum that adds above-mentioned steps 2 acquisitions of 10 times of serial dilutions (1: 100,1: 1000 and 1: 10000) is hatched altogether, and then adds the anti-rat immune globulin of alkali phosphatase enzyme mark, adds enzyme substrates at last, measures the OD value.The OD value is higher than the result that negative control group OD value adds 5 times of standard deviations and thinks the positive.Three repetitions are established in experiment, and the result is as shown in table 1, and each point is represented the mean value of 3 repeated experiments.
Table 1 antibody titers measurement result
The result shows, in all adjuvant groups, and conjugate (P64K)
2(rhEGF)
5High-level antibody in can both the inducing mouse body, aluminium adjuvant and Montanide ISA 51 do not have tangible adjuvant effect difference in the mouse body, and independent conjugate (P64K)
2(rhEGF)
5Or recombinant human epidermal growth factor mutant mixture only has lower immunogenicity in the mouse body.
Embodiment 6, conjugate (P64K)
2(rhEGF)
5Immunogenicity analysis in non-human primate
1, conjugate (P64K)
2(rhEGF)
5With Montanide ISA 51 emulsifications
Get the conjugate (P64K) of the foregoing description 3 preparations of 1.0mL oil adjuvant Montanide ISA 51 and isopyknic 1.0mg/mL with disposable sterilized pyrogen-free syringe
2(rhEGF)
5Or the recombinant human epidermal growth factor mutant mixture of isopyknic the foregoing description 1 preparation is mixed in the sample bottle, blow and beat repeatedly 20 times, make it to form water in oil mixture, the mixture after blowing and beating repeatedly is added drop-wise in the distilled water, the emulsification drop that is white in color, and do not disperse.
2, immune programme for children
Get Cercophithecus aethiop monkey and be divided into 2 groups at random, 2 every group, the adjuvant Montanide ISA51 and the conjugate (P64K) of 1 preparation of subcutaneous injection 50ug above-mentioned steps
2(rhEGF)
5Mixture or the mixture of adjuvant Montanide ISA51 and recombinant human epidermal growth factor mutant mixture, weekly immunity once, altogether immunity is 2 times.Respectively at sample of blood, drawn before the immunity, after last immune 1 month, 2 months and 3 months, separation of serum is used for antibody titers and detects.Simultaneously in contrast with the Cercophithecus aethiop monkey of injection same dose PBS.
3, antibody titers detects
Adopt the ELISA method to measure the titre of the anti-EGF antibody of specificity in the serum of above-mentioned steps 2 acquisitions.Porous plate earlier wraps quilt with EGF, add 10 times of serial dilutions (1: 10,1: 100,1: 1000 and 1: 10000) the serum that obtains of above-mentioned steps 2 hatch altogether, and then the anti-rat immune globulin of adding alkali phosphatase enzyme mark, add enzyme substrates at last, measure the OD value.The OD value is higher than the result that negative control group OD value adds 5 times of standard deviations and thinks the positive.Three repetitions, conjugate (P64K) are established in experiment
2(rhEGF)
5Immunogenicity analytical results in non-human primate as shown in figure 16, each point is represented the mean value of 3 repeated experiments.
The result shows, conjugate (P64K)
2(rhEGF)
5The highest antibody titers of immune group reaches 1: 20000, and recombinant human epidermal growth factor mutant mixture immune group antibody titers only is 1: 100.Therefore, rhEGF does not have immunogenicity in non-human primate, after recombinant human epidermal growth factor mutant mixture and the P64k coupling, can make non-human primate produce high-caliber immunne response.Show conjugate (P64K)
2(rhEGF)
5In human body, have and induce the potentiality that produce anti-EGF antibody response.
Embodiment 7, conjugate (P64K)
2(rhEGF)
5In the intravital anti-tumor activity analysis of mouse
Get 60 10 age in week BALB/C mice, the adjuvant Montanide ISA51 and the conjugate (P64K) of subcutaneous injection 0.1mL the foregoing description 5 steps 1 preparations
2(rhEGF)
5Mixture, immunity in per 7 days once, altogether immunity is 3 times.Last immunity sample of blood, drawn after 7 days, separation of serum is used for antibody titers and detects.Every mouse was transplanted 200,000 Ai Shi ascitic tumor cells (Cuba molecular immunology center provides) at the 28th day simultaneously.With the BALB/C mice of injection same dose physiological saline in contrast.In subsequently 71 days, observe the tumor growth situation of mouse, detect the level of anti-recombinant human epidermal growth factor antibody in the animal serum, the relation between assessment antibody titers, tumor growth and the survival rate the results are shown in Table 2.
Table 2 respectively organize mouse the observation period survival condition and with the corresponding relation of antibody titers
In the table 2,
*Expression does not comprise the mouse that tumor rejection occurs,
*Expression has a mouse to escape.
Survival time prolongs ratio=(X
Immune group-X
Control group)/X
Control group* 100%;
Wherein, X
Immune groupThe average survival time of expression immune group mouse, X
Control groupThe average survival time of expression control group mice.
The result shows, conjugate (P64K)
2(rhEGF)
5Immunity back inducing mouse produces anti-EGF antibody, and antibody titers is relevant with the survival of mouse, the survival rate of promptly high anti-EGF antibody capable significant prolongation tumor-bearing mice of tiring.
Sequence table
＜110〉Biotech Pharmaceutical Co., Ltd.
＜120〉a kind of conjugate and preparation method thereof and application
<130>CGGNARZ92049
<160>4
<210>1
<211>162
<212>DNA
＜213〉artificial sequence
<220>
<221>
<400>1
aactcagata?gtgaatgccc?tttgagccac?gatggatatt?gcctacatga?cggtgtttgt 60
atgtatatcg?aggctttaga?caaatacgca?tgcaactgcg?ttgttggtta?catcggagaa 120
agatgtcaat?accgtgactt?aaaatggtgg?gaattacgtt?ag 162
<210>2
<211>53
<212>PRT
＜213〉artificial sequence
<220>
<221>
<400>2
Asn?Ser?Asp?Ser?Glu?Cys?Pro?Leu?Ser?His?Asp?Gly?Tyr?Cys?Leu?His
1 5 10 15
Asp?Gly?Val?Cys?Met?Tyr?Ile?Glu?Ala?Leu?Asp?Lys?Tyr?Ala?Cys?Asn
20 25 30
Cys?Val?Val?Gly?Tyr?Ile?Gly?Glu?Arg?Cys?Gln?Tyr?Arg?Asp?Leu?Lys
35 40 45
Trp?Trp?Glu?Leu?Arg
50
<210>3
<211>1785
<212>DNA
＜213〉artificial sequence
<220>
<221>
<400>3
atggctttag?ttgaattgaa?agtgcccgac?attggcggac?acgaaaatgt?agatattatc 60
gcggttgaag?taaacgtggg?cgacactatt?gctgtggacg?ataccctgat?tactttggaa 120
accgataaag?cgactatgga?cgtacctgct?gaagttgcag?gcgtagtcaa?agaagttaaa 180
gttaaagtcg?gcgacaaaat?ctctgaaggt?ggtttgattg?tcgtcgttga?agctgaaggc 240
acggcagccg?ctcctaaagc?cgaagcggct?gccgccccgg?cgcaagaagc?ccctaaagct 300
gccgctcctg?ctccgcaagc?cgcgcaattc?ggcggttctg?ccgatgccga?gtacgacgtg 360
gtcgtattgg?gtggcggtcc?cggcggttac?tccgctgcat?ttgccgctgc?cgatgaaggc 420
ttgaaagtcg?ccatcgtcga?acgttacaaa?actttgggcg?gcgtttgcct?gaacgtcggc 480
tgtatccctt?ccaaagcctt?gttgcacaat?gccgccgtta?tcgacgaagt?gcgccacttg 540
gctgccaacg?gtatcaaata?ccccgagccg?gaactcgaca?tcgatatgct?tcgcgcctac 600
aaagacggcg?tagtttcccg?cctcacgggc?ggtttggcag?gtatggcgaa?aagccgtaaa 660
gtggacgtta?tccaaggcga?cgggcaattc?ttagatccgc?accacttgga?agtgtcgctg 720
actgccggcg?acgcgtacga?acaggcagcc?cctaccggcg?agaaaaaaat?cgttgccttc 780
aaaaactgta?tcattgcagc?aggcagccgc?gtaaccaaac?tgcctttcat?tcctgaagat 840
ccgcgcatca?tcgattccag?cggcgcattg?gctctgaaag?aagtaccggg?caaactgctg 900
attatcggcg?gcggcattat?cggcctcgag?atgggtacgg?tttacagcac?gctgggttcg 960
cgtttggatg?tggttggaat?gatggacggc?ctgatgcaag?gcgcagaccg?cgatttggta 1020
aaagtatggc?aaaaacaaaa?cgaataccgt?tttgacaaca?ttatggtcaa?caccaaaacc 1080
gttgcagttg?agccgaaaga?agacggcgtt?tacgttacct?ttgaaggcgc?gaacgcgcct 1140
aaagagccgc?aacgctacga?tgccgtattg?gttgccgccg?gccgcgcgcc?caacggcaaa 1200
ctcatcagcg?cggaaaaagc?aggcgttgcc?gtaaccgatc?gcggcttcat?cgaagtggac 1260
aaacaaatgc?gtaccaatgt?gccgcacatc?tacgccatcg?gcgacatcgt?cggtcagccg 1320
atgttggcgc?acaaagccgt?tcacgaaggc?cacgtcgccg?ccgaaaactg?cgtcggccac 1380
aaagcctact?tcgacgcgcg?cgtgattccg?ggcgttgcct?acacttcccc?cgaagtggcg 1440
tgggtgggcg?aaaccgaact?gtccgccaaa?gcctccggcc?gcaaaatcac?caaagccaac 1500
ttcccgtggg?cggcttccgg?ccgtgcgatt?gccaacggtt?gcgacaacgg?ctttaccaag 1560
ctgatttttg?atgccgaaac?cggccgcatc?atcggcggcg?gcattgtcgg?tccgaacggt 1620
ggcgatatga?tcggcgaagt?ctgccttgcc?atcgaaatgg?gctgcgacgc?ggcagacatc 1680
ggcaaaacca?tccacccgca?cccgaccttg?ggcgaatcca?tcggtatggc?ggcggaagtg 1740
gcattgggta?cttgtaccga?cctgcctccg?caaaagaaaa?aataa 1785
<210>4
<211>594
<212>PRT
＜213〉artificial sequence
<220>
<221>
<400>4
Met?Ala?Leu?Val?Glu?Leu?Lys?Val?Pro?Asp?Ile?Gly?Gly?His?Glu?Asn
1 5 10 15
Val?Asp?Ile?Ile?Ala?Val?Glu?Val?Asn?Val?Gly?Asp?Thr?Ile?Ala?Val
20 25 30
Asp?Asp?Thr?Leu?Ile?Thr?Leu?Glu?Thr?Asp?Lys?Ala?Thr?Met?Asp?Val
35 40 45
Pro?Ala?Glu?Val?Ala?Gly?Val?Val?Lys?Glu?Val?Lys?Val?Lys?Val?Gly
50 55 60
Asp?Lys?Ile?Ser?Glu?Gly?Gly?Leu?Ile?Val?Val?Val?Glu?Ala?Glu?Gly
65 70 75 80
Thr?Ala?Ala?Ala?Pro?Lys?Ala?Glu?Ala?Ala?Ala?Ala?Pro?Ala?Gln?Glu
85 90 95
Ala?Pro?Lys?Ala?Ala?Ala?Pro?Ala?Pro?Gln?Ala?Ala?Gln?Phe?Gly?Gly
100 105 110
Ser?Ala?Asp?Ala?Glu?Tyr?Asp?Val?Val?Val?Leu?Gly?Gly?Gly?Pro?Gly
115 120 125
Gly?Tyr?Ser?Ala?Ala?Phe?Ala?Ala?Ala?Asp?Glu?Gly?Leu?Lys?Val?Ala
130 135 140
Ile?Val?Glu?Arg?Tyr?Lys?Thr?Leu?Gly?Gly?Val?Cys?Leu?Asn?Val?Gly
145 150 155 160
Cys?Ile?Pro?Ser?Lys?Ala?Leu?Leu?His?Asn?Ala?Ala?Val?Ile?Asp?Glu
165 170 175
Val?Arg?His?Leu?Ala?Ala?Asn?Gly?Ile?Lys?Tyr?Pro?Glu?Pro?Glu?Leu
180 185 190
Asp?Ile?Asp?Met?Leu?Arg?Ala?Tyr?Lys?Asp?Gly?Val?Val?Ser?Arg?Leu
195 200 205
Thr?Gly?Gly?Leu?Ala?Gly?Met?Ala?Lys?Ser?Arg?Lys?Val?Asp?Val?Ile
210 215 220
Gln?Gly?Asp?Gly?Gln?Phe?Leu?Asp?Pro?His?His?Leu?Glu?Val?Ser?Leu
225 230 235 240
Thr?Ala?Gly?Asp?Ala?Tyr?Glu?Gln?Ala?Ala?Pro?Thr?Gly?Glu?Lys?Lys
245 250 255
Ile?Val?Ala?Phe?Lys?Asn?Cys?Ile?Ile?Ala?Ala?Gly?Ser?Arg?Val?Thr
260 265 270
Lys?Leu?Pro?Phe?Ile?Pro?Glu?Asp?Pro?Arg?Ile?Ile?Asp?Ser?Ser?Gly
275 280 285
Ala?Leu?Ala?Leu?Lys?Glu?Val?Pro?Gly?Lys?Leu?Leu?Ile?Ile?Gly?Gly
290 295 300
Gly?Ile?Ile?Gly?Leu?Glu?Met?Gly?Thr?Val?Tyr?Ser?Thr?Leu?Gly?Ser
305 310 315 320
Arg?Leu?Asp?Val?Val?Gly?Met?Met?Asp?Gly?Leu?Met?Gln?Gly?Ala?Asp
325 330 335
Arg?Asp?Leu?Val?Lys?Val?Trp?Gln?Lys?Gln?Asn?Glu?Tyr?Arg?Phe?Asp
340 345 350
Asn?Ile?Met?Val?Asn?Thr?Lys?Thr?Val?Ala?Val?Glu?Pro?Lys?Glu?Asp
355 360 365
Gly?Val?Tyr?Val?Thr?Phe?Glu?Gly?Ala?Asn?Ala?Pro?Lys?Glu?Pro?Gln
370 375 380
Arg?Tyr?Asp?Ala?Val?Leu?Val?Ala?Ala?Gly?Arg?Ala?Pro?Asn?Gly?Lys
385 390 395 400
Leu?Ile?Ser?Ala?Glu?Lys?Ala?Gly?Val?Ala?Val?Thr?Asp?Arg?Gly?Phe
405 410 415
Ile?Glu?Val?Asp?Lys?Gln?Met?Arg?Thr?Asn?Val?Pro?His?Ile?Tyr?Ala
420 425 430
Ile?Gly?Asp?Ile?Val?Gly?Gln?Pro?Met?Leu?Ala?His?Lys?Ala?Val?His
435 440 445
Glu?Gly?His?Val?Ala?Ala?Glu?Asn?Cys?Val?Gly?His?Lys?Ala?Tyr?Phe
450 455 460
Asp?Ala?Arg?Val?Ile?Pro?Gly?Val?Ala?Tyr?Thr?Ser?Pro?Glu?Val?Ala
465 470 475 480
Trp?Val?Gly?Glu?Thr?Glu?Leu?Ser?Ala?Lys?Ala?Ser?Gly?Arg?Lys?Ile
485 490 495
Thr?Lys?Ala?Asn?Phe?Pro?Trp?Ala?Ala?Ser?Gly?Arg?Ala?Ile?Ala?Asn
500 505 510
Gly?Cys?Asp?Asn?Gly?Phe?Thr?Lys?Leu?Ile?Phe?Asp?Ala?Glu?Thr?Gly
515 520 525
Arg?Ile?Ile?Gly?Gly?Gly?Ile?Val?Gly?Pro?Asn?Gly?Gly?Asp?Met?Ile
530 535 540
Gly?Glu?Val?Cys?Leu?Ala?Ile?Glu?Met?Gly?Cys?Asp?Ala?Ala?Asp?Ile
545 550 555 560
Gly?Lys?Thr?Ile?His?Pro?His?Pro?Thr?Leu?Gly?Glu?Ser?Ile?Gly?Met
565 570 575
Ala?Ala?Glu?Val?Ala?Leu?Gly?Thr?Cys?Thr?Asp?Leu?Pro?Pro?Gln?Lys
580 585 590
Lys?Lys
Claims (3)
1. conjugate is that mixture and the coupling of Neisseria meningitidis outer membrane protein by the recombinant human epidermal growth factor mutant obtains, and the structural formula of described conjugate is:
(P64k)
2(rhEGF)
5；
Wherein, P64K is the Neisseria meningitidis outer membrane protein, and rhEGF is the mixture of described recombinant human epidermal growth factor mutant;
The mixture of described recombinant human epidermal growth factor mutant is made up of following two kinds of albumen:
1) the 53rd of sequence 2 the albumen that the arginine disappearance obtains in the sequence table;
2) the 53rd of sequence 2 the arginine and the 52nd albumen that the leucine disappearance obtains in the sequence table.
2. vaccine that treats and/or prevents epithelium source property tumour, its activeconstituents is the described conjugate of claim 1;
Described epithelium source property tumour is nonsmall-cell lung cancer, cancer of the stomach, colorectal cancer or nasopharyngeal carcinoma.
3. vaccine according to claim 2 is characterized in that: comprise adjuvant Montanide ISA 51 or aluminium adjuvant in the described vaccine.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100770585A CN101475640B (en) | 2009-01-19 | 2009-01-19 | Conjugate, and preparation and use thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100770585A CN101475640B (en) | 2009-01-19 | 2009-01-19 | Conjugate, and preparation and use thereof |
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| CN106840811A (en) * | 2017-01-22 | 2017-06-13 | 中国科学院自动化研究所 | A kind of external pathological staining kit of tumor tissues |
| CN107064513A (en) * | 2017-01-22 | 2017-08-18 | 中国科学院自动化研究所 | A kind of tumor diagnosis kit |
| CN113855792B (en) * | 2021-12-01 | 2022-03-01 | 上海惠盾因泰生物科技有限公司 | Recombinant hEGF-CRM197 tumor therapeutic vaccine formulation |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6191106B1 (en) * | 1991-08-16 | 2001-02-20 | Chiron Corporation | Muteins of epidermal growth factor exhibiting enhanced binding at low pH |
-
2009
- 2009-01-19 CN CN2009100770585A patent/CN101475640B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6191106B1 (en) * | 1991-08-16 | 2001-02-20 | Chiron Corporation | Muteins of epidermal growth factor exhibiting enhanced binding at low pH |
Non-Patent Citations (2)
| Title |
|---|
| Elia Neninger Vinageras等.Phase II Randomized Controlled Trial of an Epidermal Growth Factor Vaccine in Advanced Non-Small-Cell Lung Cancer.Journal of Clinical Oncology.2008,26(9),1452-1458. * |
| G.González等.A novel cancer vaccine composed of human-recombinant epidermal growth factor linked to a carrier protein: Report of a pilot clinical trial.Annals of Oncology.1998,9(4),431-435. * |
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