WO2019140911A1 - <?xm-replace_text {发明名称}?> 一种阴离子多肽羧基化生物纳米磁珠及其制备方法 - Google Patents

<?xm-replace_text {发明名称}?> 一种阴离子多肽羧基化生物纳米磁珠及其制备方法 Download PDF

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WO2019140911A1
WO2019140911A1 PCT/CN2018/102326 CN2018102326W WO2019140911A1 WO 2019140911 A1 WO2019140911 A1 WO 2019140911A1 CN 2018102326 W CN2018102326 W CN 2018102326W WO 2019140911 A1 WO2019140911 A1 WO 2019140911A1
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bio
anionic polypeptide
nanomagnetic
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carboxylated
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PCT/CN2018/102326
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French (fr)
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张金菊
王红光
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北京中科圆融生物科技发展有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01F—MAGNETS; INDUCTANCES; TRANSFORMERS; SELECTION OF MATERIALS FOR THEIR MAGNETIC PROPERTIES
    • H01F1/00—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties
    • H01F1/0036—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties showing low dimensional magnetism, i.e. spin rearrangements due to a restriction of dimensions, e.g. showing giant magnetoresistivity
    • H01F1/0045—Zero dimensional, e.g. nanoparticles, soft nanoparticles for medical/biological use

Definitions

  • the invention relates to the field of nano magnetic beads application and medical detection, in particular to an anionic polypeptide carboxylated biological nano magnetic beads and a preparation method thereof.
  • Bio-nanomagnetic beads are magnetic nanoparticles produced by magnetotactic bacteria, also known as bacterial magnetic particles.
  • the inner core is Fe 3 O 4 crystal, which is coated with a layer of phospholipid biofilm and has a particle size of 30-120 nm.
  • the bio-nanomagnetic beads produced by the same kind of magnetotactic bacteria have the same particle size and crystal crystal form, uniform magnetic properties, natural biofilm coating, and good water-soluble and colloidal properties.
  • bacterial magnetic particles are a source of biological preparation and therefore have good biocompatibility.
  • the surface of the bio-nanomagnetic bead has a large number of functional groups, which can be linked to different functional macromolecules, such as antibodies, by chemical modification and bifunctional coupling agents, thereby having different special functions.
  • the most unique feature of bacterial magnetic particles is that they can express specific protein and polypeptide molecules on the surface membrane by genetic engineering methods, and become functional biological nano-magnetic beads with special biological activity.
  • Nano-magnetic particles are a kind of functional materials with a very broad application prospect, but the exposed nanoparticles are easy to aggregate, toxic to organisms and poor in biocompatibility. These shortcomings limit their wide application, especially in the biomedical field. application. Therefore, the surface of the nanomaterial is chemically modified to carry reactive functional groups (such as -COOH, -NH 2 , -OH, etc.), or to encapsulate biocompatible materials (such as oleic acid, SiO 2 , poly Ethylene glycol, etc., has always been a research hotspot and one of the important prerequisites for the application of nanoparticles in biomedical fields. Chemical coprecipitation, hydrothermal, sol-gel, microemulsion, etc.
  • reactive functional groups such as -COOH, -NH 2 , -OH, etc.
  • biocompatible materials such as oleic acid, SiO 2 , poly Ethylene glycol, etc.
  • modified nano-magnetic beads modified by the nano magnetic particles disclosed in the prior art have large differences in performance, the amount of bound antibodies is low, and the bio-nanomagnetic beads after grafting modified macromolecules have poor stability and are easily inactivated. It is difficult to promote, and the practicality is not strong.
  • the genetic modification method is relatively easy to obtain bio-nanomagnetic beads similar to chemical synthesis, and the surface film can naturally carry various functional groups, but in the preparation process of the modified bio-nano magnetic beads by genetic modification, The conditions are very strict, and the success rate of DNA transformation expression is very low, and the survival rate of the strain and the yield of the nanomagnetic beads are both low, which inhibits its wide application.
  • the present invention provides an anionic polypeptide carboxylated biological nano magnetic bead and a preparation method thereof.
  • the present invention provides an anionic polypeptide carboxylated bio-nanomagnetic bead.
  • the anionic polypeptide carboxylated bio-nanomagnetic bead is fused by a cationic linker grafted with an anionic polypeptide polymer of polyethylene glycol via a flexible linker to a membrane protein of a bio-nanomagnetic bead.
  • the amino acid residue of the flexible linker is GCVA (DLGGV) 2 GVC (GA) 3 MADEGAG.
  • G is an abbreviation for amino acid
  • Example G is an abbreviation for glycine Gly
  • C is an abbreviation for cysteine Cys.
  • the ionic polypeptide sequence is mainly composed of polyglutamic acid (PGA) or polyaspartic acid (PASP), and has the characteristics of natural glutamic acid-rich protein or synthetic ⁇ -PGA or PASP and its derivatives, respectively named YR-APE1, YR-APD2.
  • PGA polyglutamic acid
  • PASP polyaspartic acid
  • amino acid residue sequence of YR-APE1 is: AQEKNEEEETATEETEEEGAEGAEAEEEEETAEGAEDEDEDEEDGSGSQEHEEDEETEETEEGAEGEAEEAEDEAEEEDPGEEEDAQEEEEEGSGSEEEEEAQEEEDE;
  • amino acid residue sequence of YR-APD2 is: GEFDDDDDDDFDEEFDDDDDDDGDDKDDDLDGDDDDDNDGEGEGSDDDDDDDDG3DDEHHDDDGDDDHDHDDDHDDNNDDHDDDDNDHHDTDDPDHDDDHDDDDDDNNDDDNDDD;
  • the sequence of the cationic polypeptide gene was optimized for the magnetotactic bacteria MSR-I as follows:
  • the gene sequence of YR-APE1 is 5-GCCCAGGAGAAGAATGAAGAAGAAGAGACAGCCACAGAAGAGACGGAAGAAGAGGGTGCGGAAGGGGCGGAAGCCGAGGAGGAGGAAGAAACTGCAGAAGGAGCAGAAGACGAAGATGAGGATGAGGAAGATGGCTCCGGCTCCCAAGAGCATGAAGAAGATGAGGAGACTGAAGAAACAGAGGAGGGAGCAGAAGGAAGCAGAGGAAGCAGAGGACGAAGCTGAAGAAGAAGACCCAGGAGAAGAAGAAGATGCACAGGAGGAGGAA GAAGAGGGCTCCGGCTCCGAGGAAGAAGAAGAGGCCCAGGAAGAAGAGGACGAG-3;
  • the YR-APD2 gene sequence is 5- GGCGAATTTGATGACGATGATGACGATGATGATGACTTTGATGAAGAATTTGATGATGATGATGACGATGATGATGATGGCGATGATAAAGATGATGATTTAGATGGTGATGACGATGATGATAACGATGGTAGTGACGAGGGTAGTGACGACGAGGATGATGACGATGACGACGACGGCGACGACGAGCATCATGACGACGGCGACGACGACGATCCTGATCACGACGACGATCATGACGACAACAACGATGACCATGACGACGACGACAACGACCACCATGACACTGACGACCCGGACCACGACGATGACCACGACGACGACGACGACGACAATAACGCGATGATAATGACGACGACGACGAC-3.
  • the anionic polypeptide carboxylated bio-nanomagnetic beads provided by the present invention is adapted to the anionic polypeptide polymer, and the specific anionic polypeptide structure is displayed on the surface of the bio-nanomagnetic beads, and the functional group as a modification site is provided.
  • the amount of the combined antibody is large, and the magnetic activity of the magnetic beads is good and the specificity is high. It can be used as a zero-generation nano material, which is easy to carry out subsequent functional modification and multi-generation grafting of large molecules, and has high practicability.
  • anionic polypeptide carboxylated bio-nanomagnetic beads are prepared by the following steps:
  • An anionic polypeptide is prepared by a method of DNA synthesis, and an anionic polypeptide is fused with a bacterial magnetic particle membrane protein gene mamC or mamF through a flexible linker to form a gene fusion expression vector;
  • step C the expression vector obtained in step B is introduced into the primary recombinant strain obtained in step A, and the secondary recombinant strain expressing the anionic polypeptide is screened;
  • step C The second-stage recombinant strain obtained in step C is subjected to fermentation culture to produce a modified bio-nano magnetic bead expressing an anionic polypeptide;
  • the modified bio-nanomagnetic beads obtained in step D are graft-modified with a polymer glycosylated polyethylene glycol PEG to form the anionic polypeptide carboxylated bio-nanomagnetic beads having a shell structure.
  • step A in the step includes the following steps:
  • (a-1) Knockout amplifying a homologous DNA fragment of 300-700 bp long on both sides of the bacterial magnetic particle membrane protein mamC or mamF gene, and cloning the DNA fragment into phage virus AAV-del On the microcarrier, a phage-based microcarrier sequence AAV-del-mac or AAV-del-maf;
  • (a-2) Gene transfer Obtain the nucleic acid sequence product of AAV-del-mac or AAV-del-maf, adjust the concentration of the nucleic acid sequence product to 1-3 mg/ml, and transfer it to MSR-I wild strain by electroporation. in;
  • the method for constructing the gene fusion expression vector of the anionic polypeptide and mamC or mamF, the method for screening the strain, and the method for introducing the vector can all be operated by the methods disclosed in the prior art, and the present invention does not specifically explain it.
  • the anionic polypeptide carboxylated bio-nanomagnetic beads obtained by the genetic engineering method further improve the performance of the magnetic beads-bound antibody, and the obtained nano-magnetic beads have high activity, good stability, long shelf life, good environmental resistance, and high practicability. .
  • the present invention also provides a method for preparing an anionic polypeptide carboxylated biological nano magnetic bead, comprising the following steps:
  • An anionic polypeptide is prepared by a method of DNA synthesis, and an anionic polypeptide is fused with a bacterial magnetic particle membrane protein gene mamC or mamF through a flexible linker to form a gene fusion expression vector;
  • step C the expression vector obtained in step B is introduced into the primary recombinant strain obtained in step A, and the secondary recombinant strain expressing the anionic polypeptide is screened;
  • step C The second-stage recombinant strain obtained in step C is subjected to fermentation culture, and the modified bio-nano magnetic beads expressing the anionic polypeptide are isolated and purified;
  • the modified bio-nanomagnetic beads obtained in step D are graft-modified with a polymer glycosylated polyethylene glycol PEG to form the anionic polypeptide carboxylated bio-nanomagnetic beads having a shell structure.
  • the preparation of the anionic polypeptide carboxylated bio-nanomagnetic beads by the above-mentioned gene recombination method the single gene deletion of the bacterial magnetic particle membrane protein gene mamC or mamF does not affect the yield of the nano-magnetic beads, and the double mutants for constructing them are beneficial to the full
  • the protein is used as a novel fusion protein expression skeleton, and the (MamC or MamF) backbone + linker + target protein can obtain the recombinant strain better, and the obtained anionic polypeptide carboxylated biological nano magnetic beads have good activity and high stability.
  • the strain selects the MSR-I wild type strain, and the specific method of the step A includes the following steps:
  • (a-1) Knockout amplifying a homologous DNA fragment of 300-700 bp long on both sides of the bacterial magnetic particle membrane protein mamC or mamF gene, and cloning the DNA fragment into phage virus AAV-del On the microcarrier, a phage-based microcarrier sequence AAV-del-mac or AAV-del-maf;
  • (a-2) Gene transfer Obtain the nucleic acid sequence product of AAV-del-mac or AAV-del-maf, adjust the concentration of the nucleic acid sequence product to 1-3 mg/ml, and transfer it to MSR-I wild strain by electroporation. in;
  • the specific scheme of the electrical conversion is: using a square wave electric pulse, the voltage is 3100V-3200V, the electric pulse time is 3.1-3.3ms, and the number of electric pulses is 1-2 times.
  • the phage AAV-del was used as a microcarrier, and the recombinant strain was constructed by electroporation transformation.
  • the obtained recombinant strain had high survival rate, high yield and good performance.
  • step D includes the following specific steps:
  • pre-culture the secondary recombinant strain obtained in step C is inoculated into the sterilized first medium for 14-18h to obtain a pre-cultured strain, and the pre-culture condition is: temperature 35-38 ° C, ventilation The amount is 0.3-0.5 mL of gas per 1 mL of culture medium per minute, and the gas is a mixed gas of 5%-10% O 2 and 90%-95% N 2 ;
  • the obtained pre-cultured strain is inoculated into a fermenter containing the sterilized second medium for deep culture for 3-4 days to obtain a deep layer culture condition: temperature 34-37 ° C, ventilation
  • the amount is 0.4-0.6 mL of gas per 1 mL of culture medium per minute, and the gas is a mixed gas of 5% O 2 , 1% H 2 and 94% N 2 ;
  • the obtained deep culture is sequentially subjected to microbial pulverization, magnetic adsorption and gradient purification, and finally the modified bio-nanomagnetic beads expressing the anionic polypeptide are finally obtained.
  • the first medium is 1-2 parts by weight of putrescine dihydrochloride, 0.1-0.2 parts of choline chloride, 7-8 parts of D-glucose, and 1-2 parts of linoleic acid.
  • the second medium consists of 2-3 parts by weight of dextran, 2-3 parts by weight
  • the composition consists of Tween 80, 0.3-0.5 parts of thiamphenicol, 7-8 parts of D-glucose, 1-2 parts of linoleic acid, 2-3 parts of trehalose and 1-2 parts of thioglycerol.
  • the above-mentioned culture method is used to culture the secondary recombinant strain, and the growth and metabolism of the synthesized nano magnetic beads are effectively promoted.
  • the ventilation volume and the gas composition are reasonably set, and the yield of the nano magnetic beads is greatly improved.
  • step E is composed of the following sub-steps:
  • E-1, 20-30 mg of bio-nano magnetic beads are dissolved in 10-12 mL of phosphate buffer, preferably at a concentration of 0.2-0.3%, and 1-1.5 mL of 9 mM N-acetyl carboxy sugar (UDP-GalNAc) is added. Stir at room temperature for 30 min;
  • step e-2 the bio-nano magnetic beads obtained in step e-2 are separated and purified by a magnetic stand, washed, redissolved in 10-12 mL of phosphate buffer, and added with 1-2 mL of 2.5 mM sialic acid-activated PEG (CMP). -SiaPEG-20K), stir and mix; then add sialyltransferase (ST6GalNAc-I) to a final concentration of 200-280mU, shake gently at 50-80rpm at 32 ° C for 24-48 hours, catalysis Forming a bio-nano magnetic bead having a PEG polymer outer shell;
  • CMP sialic acid-activated PEG
  • step e-3 the bio-nano magnetic beads obtained in step e-3 are washed 2-3 times with 25% ethanol to obtain the bio-nano magnetic beads having a shell structure.
  • the surface of the anionic polypeptide carboxylated bio-nanomagnetic bead is further modified by the above method, and the chemical group on the surface of the natural microbial magnetic particle can be blocked, and only the linker-linked anionic polypeptide is exposed outside the core-shell structure to the biofunctional molecule. It has a good protective effect, prolongs its half-life, and further improves the stability of the bio-nano magnetic bead reagent.
  • the anionic polypeptide provided by the invention is a carboxylated bio-nanomagnetic bead.
  • the anionic polypeptide is attached to the membrane protein of the nano-magnetic bead through a specific suitable linker, and the three are well-bonded, and the number of binding carboxyl-based sites is increased.
  • the amount of antibody is high, 1mg nano-magnetic beads can bind more than 120 ⁇ g of antibody, and has good practicability; at the same time, it has high stability and good environmental tolerance, and still maintains about 75% of original activity after 14 days at 37 °C. It has a valid period of more than one and a half years under conventional storage conditions and has good economic benefits.
  • the method for preparing an anionic polypeptide carboxylated biological nano magnetic bead provided by the invention has a survival rate of more than 80%, and a success rate of DNA transformation expression is higher than 60%, and the obtained bio-nano magnetic beads have a large amount and can be widely applied.
  • An anionic polypeptide carboxylated bio-nanomagnetic bead which is formed by fusion polymerization of an anionic polypeptide polymer grafted with polyethylene glycol through a flexible linker to a membrane protein of a bio-nanomagnetic bead, and the amino acid residue of the flexible linker is GCVA (DLGGV) 2 GVC (GA) 3 MADEGAG; the amino acid residue of the anionic polypeptide polymer is AQEKNEEEETATEETEEEGAEGAEAEEEEETAEGAEDEDEDEDEDGSGSQEHEEDEETEETEEGAEGEAEEAEDEAEEEDPGEEEDAQEEEEEGSGSEEEEEAQEEEDE.
  • An anionic polypeptide carboxylated bio-nanomagnetic bead which is formed by fusion polymerization of an anionic polypeptide polymer grafted with polyethylene glycol through a flexible linker to a membrane protein of a bio-nanomagnetic bead, and the amino acid residue of the flexible linker is GCVA (DLGGV) 2 GVC (GA) 3 MADEGAG; the amino acid residue of the anionic polypeptide polymer is GEFDDDDDDDFDEEFDDDDDDDGDDKDDDLDGDDDDDGSGSGSEDEDDDDDGDDEHHDDDGDDDHDHDDDHDDNNDDHDDDDNDHHDTDDPDHDDDHDDDDDDNNDDDDDDDDD.
  • An anionic polypeptide carboxylated bio-nanomagnetic bead which is formed by fusion polymerization of an anionic polypeptide polymer grafted with polyethylene glycol through a flexible linker to a membrane protein of a bio-nanomagnetic bead, and the amino acid residue of the flexible linker is GCVA (DLGGV) 2 GVC (GA) 3 MADEGAG; the amino acid residue of the anionic polypeptide polymer is AQEKNEEEETATEETEEEGAEGAEAEEEEETAEGAEDEDEDEDEDGSGSQEHEEDEETEETEEGAEGEAEEAEDEAEEEDPGEEEDAQEEEEEGSGSEEEEEAQEEEDE.
  • An anionic polypeptide is prepared by a method of DNA synthesis, and an anionic polypeptide is fused with a bacterial magnetic particle membrane protein gene mamC or mamF through a flexible linker to form a gene fusion expression vector;
  • step C the expression vector obtained in step B is introduced into the primary recombinant strain obtained in step A, and the secondary recombinant strain expressing the anionic polypeptide is screened;
  • step C The second-stage recombinant strain obtained in step C is subjected to fermentation culture to produce a modified bio-nano magnetic bead expressing an anionic polypeptide;
  • the modified bio-nanomagnetic beads obtained in step D are graft-modified with a polymer glycosylated polyethylene glycol PEG to form the anionic polypeptide carboxylated bio-nanomagnetic beads having a shell structure.
  • a method for preparing a mutant strain of a membrane protein gene mamC or mamF deletion a method for preparing a gene fusion expression vector, introduction of an expression vector, screening of a strain, fermentation culture of a strain, and obtaining a biological nano magnetic bead from a strain
  • the method can be implemented by using the prior art, and is not specifically limited in this embodiment.
  • a method for preparing an anionic polypeptide carboxylated bio-nanomagnetic bead as provided in Example 1, wherein the strain selects a MSR-I wild-type strain comprising the following steps:
  • (a-2) gene transfer obtaining the nucleic acid sequence product of AAV-del-mac, adjusting the concentration of the nucleic acid sequence product to 1 mg/ml, and transferring it into the MSR-I wild strain by electroporation; using square wave electric pulse, The voltage is 3100V, the electrical pulse time is 3.1ms, and the number of electrical pulses is 2 times;
  • An anionic polypeptide is prepared by a method of DNA synthesis, and an anionic polypeptide is fused with a bacterial magnetic particle membrane protein gene mamC through a flexible linker to form a gene fusion expression vector;
  • the expression vector obtained in the step B is introduced into the primary recombinant strain MSRI-dC obtained in the step A, and the secondary recombinant strain expressing the anionic polypeptide is selected;
  • step C The second-stage recombinant strain obtained in step C is subjected to fermentation culture, and the modified bio-nano magnetic beads expressing the anionic polypeptide are isolated and purified;
  • the modified bio-nanomagnetic beads obtained in step D are graft-modified with a polymer glycosylated polyethylene glycol PEG to form the anionic polypeptide carboxylated bio-nanomagnetic beads having a shell structure.
  • a method for preparing an anionic polypeptide carboxylated bio-nanomagnetic bead as provided in Example 1 and Example 2, wherein the strain selects a MSR-I wild-type strain comprising the following steps:
  • (a-2) Gene transfer The nucleic acid sequence product of AAV-del-maf was obtained by plasmid extraction and restriction enzyme digestion, and the concentration of the nucleic acid sequence product was adjusted to 3 mg/ml, and transferred into the MSR-I wild strain by electroporation. Using a square wave electric pulse, the voltage is 3200V, the electrical pulse time is 3.3ms, and the number of electrical pulses is 1 time;
  • anionic polypeptides YR-APE1 and YR-APD2 were prepared by DNA synthesis.
  • the anionic polypeptides YR-APE1 and YR-APD2 were fused with the bacterial magnetic particle membrane protein gene mamF by a flexible linker to form a gene fusion expression vector.
  • pmamF-APE1 and pmamF-APD2 were cloned into the expression vector pBRC, and two new fusion gene fragments, pBRC-pmamF-APE1 and pBRC-pmamF-APD2, were obtained, respectively.
  • pBRC-pmamF-APE1, pBRC-pmamF-APD2 were transferred into the primary recombinant strain MSRI-dF, and the recombinant strains expressing different anionic polypeptides, ie, the secondary recombinant strains, were identified as MSRI-dF. /APE1, MSRI-dF/APD2;
  • step C The second-stage recombinant strain obtained in step C is subjected to fermentation culture, and the modified bio-nano magnetic beads expressing the anionic polypeptide are isolated and purified;
  • the modified bio-nanomagnetic beads obtained in step D are graft-modified with a polymer glycosylated polyethylene glycol PEG to form the anionic polypeptide carboxylated bio-nanomagnetic beads having a shell structure.
  • a method for preparing an anionic polypeptide carboxylated bio-nanomagnetic bead comprises the following steps:
  • (a-2) gene transfer obtaining the nucleic acid sequence product of AAV-del-mac, adjusting the concentration of the nucleic acid sequence product to 2 mg/ml, and transferring it into the MSR-I wild strain by electroporation; using square wave electric pulse, The voltage is 3100V, the electrical pulse time is 3.2ms, and the number of electrical pulses is 2 times;
  • the expression sequence of the anionic polypeptide YR-APE1 was prepared by DNA synthesis, and the anionic polypeptide YR-APE1 and the bacterial magnetic particle membrane protein gene mamC were fused by a flexible linker to form a gene fusion expression vector pmamC-APE1;
  • pre-culture the secondary recombinant strain obtained in step C is inoculated into the sterilized first medium for 14 hours to obtain a pre-cultured strain, the pre-culture condition is: temperature 35 ° C, ventilation is per minute 0.5 mL of gas is introduced per 1 mL of the medium, and the gas is a mixed gas of 5% O 2 and 95% N 2 ;
  • the obtained pre-cultured strain was inoculated into a fermenter containing the sterilized second medium for deep culture for 3 days to obtain a deep layer culture condition: the temperature was 37 ° C, and the aeration amount was per minute.
  • 0.4 mL of gas is introduced per 1 mL of the medium, and the gas is a mixed gas of 5% O 2 , 1% H 2 and 94% N 2 ;
  • the obtained deep culture is sequentially subjected to cell pulverization, magnetic adsorption and gradient purification steps, and finally the modified bio-nano magnetic beads expressing the anionic polypeptide are obtained;
  • the modified bio-nanomagnetic beads obtained in the step d-3 are graft-modified by a multi-glycosylated polyethylene glycol PEG to form the anionic polypeptide carboxylated bio-nanomagnetic beads having a shell structure.
  • step D comprises the following specific steps:
  • pre-culture the secondary recombinant strain obtained in step C is inoculated into the sterilized first medium for 18 hours to obtain a pre-cultured strain, the pre-culture condition is: temperature 37 ° C, ventilation is per minute 0.3 mL of gas is introduced per 1 mL of the medium, and the gas is a mixed gas of 10% O 2 and 90% N 2 ;
  • the obtained pre-cultured strain was inoculated into a fermentor containing the sterilized second medium for deep culture for 4 days to obtain a deep layer culture condition: a temperature of 37 ° C, and a ventilation amount per minute 0.6 mL of gas is introduced per 1 mL of the medium, and the gas is a mixed gas of 5% O 2 , 1% H 2 and 94% N 2 ;
  • the obtained deep culture is sequentially subjected to pulverization, magnetic adsorption and gradient purification steps to finally obtain the modified bio-nanomagnetic beads expressing the anionic polypeptide.
  • step D comprises the following specific steps:
  • pre-culture the secondary recombinant strain obtained in step C is inoculated into the sterilized first medium for 16h to obtain a pre-cultured strain.
  • the pre-culture condition is: temperature 38 ° C, ventilation is per minute 0.4 mL of gas was introduced per 1 mL of the medium, and the gas was a mixed gas of 7% O 2 and 93% N 2 .
  • the obtained pre-cultured strain was inoculated into a fermentor containing the sterilized second medium for deep culture for 4 days to obtain a deep layer culture condition: the temperature was 34 ° C, and the aeration amount was per minute.
  • 0.5 mL of gas is introduced per 1 mL of the medium, and the gas is a mixed gas of 5% O 2 , 1% H 2 and 94% N 2 ;
  • the obtained deep culture is sequentially subjected to pulverization, magnetic adsorption and gradient purification steps to finally obtain the modified bio-nanomagnetic beads expressing the anionic polypeptide.
  • the first medium consists of 1 part by weight of putrescine dihydrochloride, 0.2 parts of choline chloride, 8 parts of D-glucose, 2 parts of linoleic acid, 2 parts of thioglycerol, 1 part of sodium acetate. 3 agar and 2 parts ammonium alginate; the second medium consists of 3 parts by weight of dextran, 2 parts of Tween 80, 0.5 parts of thiamphenicol, 8 parts of D-glucose, 2 parts of linoleic acid 3 parts of trehalose and 1 part of thioglycerol.
  • a method for preparing an anionic polypeptide carboxylated bio-nanomagnetic bead which differs from Example 8 in that the first medium consists of 2 parts by weight of putrescine dihydrochloride, 0.1 part of choline chloride, 7 parts of D-glucose, 1 part of linoleic acid, 3 parts of thioglycerol, 0.5 part of sodium acetate, 5 agar and 1 part of ammonium alginate; the second medium consists of 2 parts by weight of dextran, 3 parts by weight Tween 80, 0.3 parts of thiamphenicol, 7 parts of D-glucose, 1 part of linoleic acid, 2 parts of trehalose and 2 parts of thioglycerol.
  • step E comprises the following specific steps:
  • the bio-nano magnetic beads obtained in step e-2 are separated and purified by a magnetic stand. After washing, redissolve in 10 mL of phosphate buffer, add 1 mL of sialic acid activated PEG at a concentration of 2.5 mM, stir and mix; Adding sialyltransferase to a final concentration of 200 mU, shaking the reaction at 80 rpm for 24 hours at 32 ° C, catalyzing the formation of bio-nanomagnetic beads with a PEG polymer shell;
  • the bio-nano magnetic beads obtained in the step e-3 are washed twice with 25% ethanol to obtain the anion polypeptide carboxylated bio-nanomagnetic beads having a shell structure.
  • step E comprises the following specific steps:
  • step e-2 the bio-nano magnetic beads obtained in step e-2 are separated and purified by a magnetic stand, washed, redissolved in 12 mL of phosphate buffer, 2 mL of sialic acid activated PEG at a concentration of 2.5 mM, stirred and mixed; Add sialyltransferase to a final concentration of 280 mU, gently shake at 48 rpm for 48 hours at 32 ° C to catalyze the formation of bio-nanomagnetic beads with a PEG polymer shell;
  • the bio-nano magnetic beads obtained in the step e-3 are washed three times with 25% ethanol to obtain the anion polypeptide carboxylated bio-nanomagnetic beads having a shell structure.
  • step E comprises the following specific steps:
  • E-1 taking 25mg of bio-nano magnetic beads dissolved in 10mL phosphate buffer, preferably 0.3%, adding 1mL of 9 mM N-acetyl carboxy sugar, stirring at room temperature for 30min;
  • the bio-nano magnetic beads obtained in step e-2 are separated and purified by a magnetic stand. After washing, redissolve in 10 mL of phosphate buffer, add 1 mL of sialic acid activated PEG at a concentration of 2.5 mM, stir and mix; Adding sialyltransferase to a final concentration of 250 mU, shaking the reaction at 32 rpm for 30 hours at a speed of 70 rpm, catalyzing the formation of bio-nanomagnetic beads with a PEG polymer shell;
  • the bio-nano magnetic beads obtained in the step e-3 are washed three times with 25% ethanol to obtain the anion polypeptide carboxylated bio-nanomagnetic beads having a shell structure.
  • An anionic polypeptide carboxylated bio-nanomagnetic bead which is formed by fusion polymerization of an anionic polypeptide polymer grafted with polyethylene glycol through a flexible linker to a membrane protein of a bio-nanomagnetic bead, and the amino acid residue of the flexible linker is GCVADLGGVGVCGAMADEGAG; the amino acid residue of the anionic polypeptide polymer is AQEKNEEEETATEETEEEGAEGAEAEEEEETAEGAEDEDEDEDEDGSGSQEHEEDEETEETEEGAEGEAEEAEDEAEEEDPGEEEDAQEEEEEGSGSEEEEEAQEEEDE.
  • An anionic polypeptide carboxylated bio-nanomagnetic bead which is formed by fusion polymerization of an anionic polypeptide polymer grafted with polyethylene glycol through a flexible linker to a membrane protein of a bio-nanomagnetic bead, and the amino acid residue of the flexible linker is GCVADLGAGAMGVGVCGGVDLGGAADEGAG; the amino acid residue of the anionic polypeptide polymer is AQEKNEEEETATEETEEEGAEGAEAEEEEETAEGAEDEDEDEDEDGSGSQEHEEDEETEETEEGAEGEAEEAEDEAEEEDPGEEEDAQEEEEEGSGSEEEEEAQEEEDE.
  • An anionic polypeptide carboxylated bio-nanomagnetic bead which is formed by fusion polymerization of an anionic polypeptide polymer grafted with polyethylene glycol through a flexible linker to a membrane protein of a bio-nanomagnetic bead, and the amino acid residue of the flexible linker is GCVA (DLGGV) 2 GVC (GA) 3 M (ADEGAG) 2 ;
  • the amino acid residue of the anionic polypeptide polymer is AQEKNEEEETATEETEEEGAEGAEAEEEEETAEGAEDEDEDEDEDGSGSQEHEEDEETEETEEGAEGEAEEAEDEAEEEDPGEEEDAQEEEEEGSGSEEEEEAQEEEDE.
  • An anionic polypeptide carboxylated bio-nanomagnetic bead which is formed by fusion polymerization of an anionic polypeptide polymer grafted with polyethylene glycol through a flexible linker to a membrane protein of a bio-nanomagnetic bead, and the amino acid residue of the flexible linker is GCVA (DLGGV) 2 GVC (GA) 3 M (ADEGAG) 2 ;
  • the amino acid residue of the anionic polypeptide polymer is AKNEEQEEETATEETEEEGAEAEEEGAEEEDEEDGSGSQETADEEGAEDEDEETHEEEETEEGAEGEAEEGEEEDAQEEEAEDEAEEEDPEEEGSGSEEEEAEEQEDE.
  • the nucleic acid sequence product of -mac is transferred into the MSR-I wild strain by parental binding.
  • Example 1 The method for preparing the anionic polypeptide carboxylated bio-nanomagnetic beads as provided in Example 1 differs from Example 4 in that the specific parameters of the electrotransformation are shown in Table 1.
  • Example 2 The method for preparing the anionic polypeptide carboxylated bio-nanomagnetic beads as provided in Example 1 differs from Example 7 in that the specific parameters of the culture are shown in Table 2.
  • Each group of bio-nano magnetic beads was magnetically adsorbed, and then the water was weighed to weigh the weight M0, and the magnetic beads were resuspended by adding the preservation solution to make the concentration of the magnetic beads 1 mg/mL.
  • FITC-Ab-EDC antibody Purchase the FITC-Ab-EDC antibody, adjust the concentration to 1 mg/mL, serially dilute in a gradient of 1/10, calculate the fluorescence intensity of each gradient by a fluorescence analyzer, and prepare a fluorescence intensity standard curve;
  • FITC-Ab-EDC antibody Take 0.5mL of FITC-Ab-EDC antibody at a concentration of 0.1mg/mL, add 100 ⁇ L nano-magnetic beads, mix well, incubate at 37 °C for 15min, mix 3-5 times during this time; magnetically adsorb magnetic beads, absorb the supernatant The FITC fluorescence intensity of the supernatant is detected, and the magnetic beads are washed several times at the same time, the antibody with weak binding is removed, and the fluorescence intensity in the solution is detected after resuspending the magnetic beads;
  • the amount of antibody corresponding to the fluorescence intensity can be calculated by the standard curve, and the loading of the antibody on the actual label of the nano-magnetic bead is measured by indirect and direct methods.
  • the results are shown in Table 3, wherein the amount of bound antibody is 1 mg of bio-nanobeads. The amount of antibody.
  • the anionic polypeptide carboxylated bio-nanomagnetic beads provided by the present invention and the anionic polypeptide are linked to the membrane protein of the nano magnetic beads through a specific suitable linker, and the three are mutually well-bonded, and the resulting carboxyl group can be combined.
  • the number of sites is large, the amount of bound antibody is high, and the utility is good; wherein the bio-nanomagnetic beads prepared by the method provided in Example 4 have the highest binding amount and the best performance.
  • the bio-nanomagnetic beads provided in Comparative Examples 1-4 were examined for the flexible Linker and the bound proteins.
  • Test Example 2 Stability test of anionic polypeptide carboxylated bio-nanomagnetic beads (high temperature accelerated test)
  • the FITC-Ab-EDC antibody-conjugated bio-nanomagnetic beads obtained in Test Example 1 and Comparative Examples 1-11 were divided into 14 groups, and each group was used at 37 ° C for each group per day.
  • One tube was taken out, washed several times with PBS, resuspended after magnetic adsorption, and the FITC fluorescence intensity was measured. Each time, it was compared with the fluorescence intensity of the reagent stored at normal 4 ° C, and finally the attenuation curve of the fluorescence intensity of the nano magnetic bead reagent was obtained.
  • the attenuation is >35% as the internal standard of the enterprise, and the storage time of the reagent failure is set. The results are shown in Table 4.
  • the bio-nanomagnetic beads provided by the present invention can still maintain about 60% of the original activity in 14 days, and the bio-nano magnetic beads obtained by the preparation methods of Examples 3 and 4 are After 14 days, the original activity was still about 75%, while the bio-nano bead provided by the control example was inferior in stability; the bio-nano magnetic beads provided by the invention have high stability, good tolerance to the environment, and good Economic benefits.
  • bio-nanomagnetic beads obtained in the preparation methods of Examples 4, 7, and 8 and Comparative Examples 5 to 16 were tested.
  • the success rate and the survival rate of the strain could not be optimal at the same time; while the comparative examples 12-16 were compared with the example 7, the gas introduction amount and the incoming components during the fermentation culture were found, and the survival of the strain, the nano magnetic beads
  • the yield has a great influence, and the invention has outstanding practical effects in improving the yield of nano magnetic beads by improving the gas introduced during the fermentation process.

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Abstract

一种阴离子多肽羧基化生物纳米磁珠，由阴离子多肽聚合物通过柔性linker连接到生物纳米磁珠的膜蛋白上融合表达而成，所述柔性linker的氨基酸残基为GCVA(DLGGV) 2GVC(GA) 3MADEGAG，在生物纳米磁珠表面展示特定阴离子多肽结构，提供的作为修饰位点的功能基团数量多，结合抗体量大，磁珠的生物活性好，特异性较高，实用性好。

Description

一种阴离子多肽羧基化生物纳米磁珠及其制备方法 技术领域
本发明涉及纳米磁珠应用和医学检测领域，具体是涉及阴离子多肽羧基化生物纳米磁珠及其制备方法。
背景技术
生物纳米磁珠是趋磁细菌生产的一种磁性纳米颗粒，也称为细菌磁颗粒，内核是Fe3O4晶体，外面有一层磷脂生物膜包被，粒径在30-120nm之间。同一种趋磁细菌生产的生物纳米磁珠，它们的粒径大小和晶体晶型基本一致，磁学性质均一，有天然生物膜包被，具有很好的水溶性质和胶体性质。此外，细菌磁颗粒是生物制备来源，因此具有较好的生物相容性。生物纳米磁珠表面膜上带有大量的功能基团，可通过化学修饰和双功能偶联剂连接不同的功能大分子，如抗体，从而具有不同的特殊功能。细菌磁颗粒最独特的地方在于它可以通过基因工程的方法在表面膜上表达特殊的蛋白质及多肽分子，成为具有特殊生物活性的功能性生物纳米磁珠。
 技术问题
纳米磁性颗粒是应用前景非常广阔的一类功能材料，但是裸露的纳米颗粒容易集聚、对生物体具有毒性及生物相容性较差，这些缺点限制了其广泛应用，尤其是在生物医学领域的应用。因此，对纳米材料进行表面的化学修饰，使其带上反应性功能基团（如-COOH，-NH2，-OH等），或者包裹生物相容性材料（如油酸、SiO2，聚乙二醇等），一直是研究的热点以及纳米颗粒应用于生物医学领域的重要前提条件之一。化学共沉淀法、水热法、溶胶-凝胶法、微乳液法等一直是化学制备纳米材料并在表面修饰生物相容性物质的常用方法，已经实现通过多代嫁接的方法，在纳米材料表面修饰树状大分子的多聚物。但现有技术中公开的纳米磁性颗粒修饰后的改性生物纳米磁珠性能差异较大，结合抗体量较低，且嫁接修饰大分子后的生物纳米磁珠稳定性较差，非常容易失活，导致难以推广，实用性不强。另一方面，基因改造方法较容易的获得类似化学合成的生物纳米磁珠，表面膜上可以天然带有各种功能基团，但在通过基因改造对于改性生物纳米磁珠的制备过程中，对条件要求非常严格，且DNA转化表达成功率非常低，菌株成活率和纳米磁珠的产率均较低，抑制其广泛的应用。
技术解决方案
为了解决上述技术问题，本发明提供一种阴离子多肽羧基化生物纳米磁珠及其制备方法。
本发明具体技术方案如下：
本发明提供一种阴离子多肽羧基化生物纳米磁珠，所述阴离子多肽羧基化生物纳米磁珠由嫁接有聚乙二醇的阴离子多肽聚合物通过柔性linker连接到生物纳米磁珠的膜蛋白上融合表达而成，所述柔性linker的氨基酸残基为GCVA(DLGGV)2GVC(GA)3MADEGAG。其中G等字母均为氨基酸的缩写，例G为甘氨酸Gly的缩写，C为半胱氨酸Cys的缩写等。
进一步地，离子多肽序列以聚谷氨酸（PGA）或聚天冬氨酸（PASP）为主，具有天然富谷氨酸蛋白或人工合成γ-PGA或PASP及其衍生物的特点，分别命名为YR-APE1，YR-APD2。
YR-APE1的氨基酸残基序列为：AQEKNEEEETATEETEEEGAEGAEAEEEEETAEGAEDEDEDEEDGSGSQEHEEDEETEETEEGAEGEAEEAEDEAEEEDPGEEEDAQEEEEEGSGSEEEEEAQEEEDE；
YR-APD2的氨基酸残基序列为：GEFDDDDDDDDDFDEEFDDDDDDDDDGDDKDDDLDGDDDDDNDGSDEGSDDEDDDDDDDG3DDEHHDDDGDDDPDHDDDHDDNNDDHDDDDNDHHDTDDPDHDDDHDDDDDDNNDDDNDDDDD；
针对趋磁细菌MSR-Ⅰ对阳离子多肽基因序列优化如下：
YR-APE1的基因序列为5-GCCCAGGAGAAGAATGAAGAAGAAGAGACAGCCACAGAAGAGACGGAAGAAGAGGGTGCGGAAGGGGCGGAAGCCGAGGAGGAGGAAGAAACTGCAGAAGGAGCAGAAGACGAAGATGAGGATGAGGAAGATGGCTCCGGCTCCCAAGAGCATGAAGAAGATGAGGAGACTGAAGAAACAGAGGAGGGAGCAGAAGGAGAAGCAGAGGAAGCAGAGGACGAAGCTGAAGAAGAAGACCCAGGAGAAGAAGAAGATGCACAGGAGGAGGAA GAAGAGGGCTCCGGCTCCGAGGAAGAAGAAGAGGCCCAGGAAGAAGAGGACGAG-3；
YR-APD2基因序列为5- GGCGAATTTGATGACGATGATGACGATGATGATGACTTTGATGAAGAATTTGATGATGATGATGACGATGATGATGATGGCGATGATAAAGATGATGATTTAGATGGTGATGACGATGATGATAACGATGGTAGTGACGAGGGTAGTGACGACGAGGATGATGACGATGACGACGACGGCGACGACGAGCATCATGACGACGACGGCGACGACGATCCTGATCACGACGACGATCATGACGACAACAACGATGACCATGACGACGACGACAACGACCACCATGACACTGACGACCCGGACCACGACGATGACCACGACGACGACGACGACGACAATAACGCGATGATAATGACGACGACGACGAC-3。
本发明所提供的阴离子多肽羧基化生物纳米磁珠，给定的柔性linker于阴离子多肽聚合物相适配，在生物纳米磁珠表面展示特定阴离子多肽结构，提供的作为修饰位点的功能基团数量多，结合抗体量大，磁珠的生物活性好，特异性较高。且可以作为零代纳米材料，易于进行后续的其他功能修饰和大分子多代嫁接，实用性高。
进一步地，所述阴离子多肽羧基化生物纳米磁珠由如下步骤制备而成：
A、构建细菌磁颗粒膜蛋白基因mamC或mamF缺失的突变体菌株，得到一级重组菌株；
B、通过DNA合成的方法制备阴离子多肽，将阴离子多肽与细菌磁颗粒膜蛋白基因mamC或mamF通过柔性linker进行融合形成基因融合表达载体；
C、将步骤B得到的表达载体导入到步骤A得到的一级重组菌株中，筛选出表达阴离子多肽的二级重组菌株；
D、将步骤C得到的二级重组菌种进行发酵培养，生产表达展示阴离子多肽的改性生物纳米磁珠；
E、采用多聚物糖基化聚乙二醇PEG对步骤D所得到的改性生物纳米磁珠进行嫁接修饰形成具有壳结构的所述阴离子多肽羧基化生物纳米磁珠。
优选地，所述步骤所述步骤A的具体方法包括如下步骤：
（a-1）基因敲除：扩增细菌磁颗粒膜蛋白mamC或mamF基因左右两侧共两个长均为300-700bp的同源DNA片段，将所述DNA片段克隆在噬菌体病毒AAV-del微载体上，即得一条基于噬菌体病毒的微载体序列AAV-del-mac或AAV-del-maf；
（a-2）基因转移：获取AAV-del-mac或AAV-del-maf的核酸序列产物，调节核酸序列产物的浓度为1-3mg/ml，通过电转化的方式转入MSR-Ⅰ野生菌株中；
（a-3）菌株筛选：经过梯度筛选，获得mamc或mamf缺失突变的重组菌株，验证后即得一级重组菌株MSRⅠ-dC。
阴离子多肽与mamC或mamF的基因融合表达载体的构建方法、筛选菌种的方法、载体导入的方法均可以通过现有技术所公开的方法进行操作，本发明不做具体解释。
采用基因工程改造方法得到的阴离子多肽羧基化生物纳米磁珠，进一步提高了磁珠结合抗体性能，且得到的纳米磁珠活性高，稳定性好，保质期长，抗环境影响能力好，实用性强。
另一方面，本发明还提供了一种阴离子多肽羧基化生物纳米磁珠的制备方法，包括如下步骤：
A、构建细菌磁颗粒膜蛋白基因mamC或mamF缺失的突变体菌株，得到一级重组菌株；
B、通过DNA合成的方法制备阴离子多肽，将阴离子多肽与细菌磁颗粒膜蛋白基因mamC或mamF通过柔性linker进行融合形成基因融合表达载体；
C、将步骤B得到的表达载体导入到步骤A得到的一级重组菌株中，筛选出表达阴离子多肽的二级重组菌株；
D、将步骤C得到的二级重组菌种进行发酵培养，分离纯化生产表达展示阴离子多肽的改性生物纳米磁珠；
E、采用多聚物糖基化聚乙二醇PEG对步骤D所得到的改性生物纳米磁珠进行嫁接修饰形成具有壳结构的所述阴离子多肽羧基化生物纳米磁珠。
采用上述基因重组的方法进行阴离子多肽羧基化生物纳米磁珠的制备，细菌磁颗粒膜蛋白基因mamC或mamF的单基因缺失对纳米磁珠产量不会产生影响，构建它们的双突变体有利于充分发挥这个蛋白作为新融合蛋白表达骨架的作用，（MamC或MamF）骨架+linker+目的蛋白可以更好的拿到重组菌种，得到的阴离子多肽羧基化生物纳米磁珠活性好，稳定性高。
进一步地，所述菌株选择MSR-Ⅰ野生型菌株，所述步骤A的具体方法包括如下步骤：
（a-1）基因敲除：扩增细菌磁颗粒膜蛋白mamC或mamF基因左右两侧共两个长均为300-700bp的同源DNA片段，将所述DNA片段克隆在噬菌体病毒AAV-del微载体上，即得一条基于噬菌体病毒的微载体序列AAV-del-mac或AAV-del-maf；
（a-2）基因转移：获取AAV-del-mac或AAV-del-maf的核酸序列产物，调节核酸序列产物的浓度为1-3mg/ml，通过电转化的方式转入MSR-Ⅰ野生菌株中；
（a-3）菌株筛选：经过梯度筛选，获得mamc或mamf缺失突变的重组菌株，验证后即得一级重组菌株MSRⅠ-dC或MSRⅠ-dF。
优选地，所述电转化的具体方案为：采用方波电脉冲，电压为3100V-3200V，电脉冲时间是3.1-3.3ms，电脉冲次数是1-2次。
采用噬菌体AAV-del作为微载体，电穿孔转化的方式进行一级重组菌株的构建，得到的重组菌株成活率高，产量较高，性能好；
进一步地，所述步骤D包括如下具体步骤：
d-1、预培养：将步骤C得到的二级重组菌株接种到经过灭菌后的第一培养基中培养14-18h后得到预培养菌株，预培养条件为：温度35-38℃，通气量为每分钟每1mL培养基通入0.3-0.5mL的气体，所述气体为5%-10%O2和90%-95%N2的混合气体；
d-2、将所得的预培养菌株接种至装有灭菌第二培养基的发酵罐中进行深层培养3-4天后得到深层培养物，所述深层培养条件为：温度34-37℃，通气量为每分钟每1mL培养基通入0.4-0.6mL的气体，所述气体为5%O2、1%H2和94%N2的混合气体；
d-3、将得到的深层培养物依次进行菌体粉碎、磁力吸附和梯度纯化不步骤，最终得到所述表达展示阴离子多肽的改性生物纳米磁珠。
优选地，所述第一培养基由重量份数为1-2份腐胺二盐酸盐、0.1-0.2份多氯化胆碱、7-8份D-葡萄糖、1-2份亚油酸、2-3份硫代甘油、0.5-1份乙酸钠、3-5琼脂和1-2份海藻酸铵组成；所述第二培养基由重量份数为2-3份右旋糖酐、2-3份吐温80、0.3-0.5份甲砜霉素、7-8份D-葡萄糖、1-2份亚油酸、2-3份海藻糖和1-2份硫代甘油组成。
采用上述培养方法对二级重组菌株进行培养，有效促进合成纳米磁珠的生长代谢，在发酵培养过程中，合理设置通气量和气体成分，大大提高了纳米磁珠的产量。
更进一步地，所述步骤E由如下分步骤构成：
e-1、取20-30mg的生物纳米磁珠溶于10-12mL磷酸缓冲液中，优选浓度为0.2-0.3%，加入1-1.5mL浓度为9mM的N乙酰羧基糖（UDP-GalNAc），室温搅拌30min；
e-2、加入N乙酰羧基糖转移酶（GalNAc-T2），至终浓度35-45mU，室温搅拌3-5h，得到UDP-GalNAc定点修饰的生物纳米磁珠；
e-3、将步骤e-2得到的生物纳米磁珠采用磁力架分离纯化，洗涤后，重新溶于10-12mL磷酸缓冲液中，加入1-2mL浓度为2.5mM的唾液酸活化PEG（CMP-SiaPEG-20K），搅拌混匀；然后加入唾液酸转移酶（ST6GalNAc-I），至终浓度200-280mU，于32℃，以50-80rpm的速度，轻轻摇晃反应24-48小时，催化形成具有PEG聚合物外壳的生物纳米磁珠；
e-4、将步骤e-3得到的生物纳米磁珠用25%乙醇洗涤2-3次，即得所述具有壳结构的生物纳米磁珠。
采用上述方法对阴离子多肽羧基化生物纳米磁珠的表面进行进一步修饰，可以将天然微生物磁颗粒表面的化学基团进行封闭，仅暴露linker连接的阴离子多肽在核壳结构之外，对生物功能分子有很好的保护效果，延长其半衰期，进一步提高生物纳米磁珠试剂的稳定性。
 有益效果
本发明所提供的阴离子多肽羧基化生物纳米磁珠，阴离子多肽通过特定适宜的linker连接在纳米磁珠的膜蛋白上，三者相互结合性好，产生的可结合羧基基位点数量多，结合抗体量高，1mg纳米磁珠可以结合120μg以上的抗体，实用性好；同时具有稳定性高，环境耐受性好的特点，在37℃条件下在14天后依旧保持原有活性的75%左右，在常规保存条件下有效期超过一年半，具有较好的经济效益。本发明所提供的制备阴离子多肽羧基化生物那纳米磁珠的方法，菌株成活率高于80%，同时DNA转化表达成功率高于60%，得到的生物纳米磁珠量大，可以广泛应用。
 本发明的实施方式
下面将对本发明的技术方案进行清楚、完整的描述，显然，所描述的实施例仅是本发明的一部分实施例，不能用来限制本发明的范围。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其它实施例，都属于本发明保护的范围，本发明没有做具体限定的技术特征均可以通过现有技术公开的常规技术进行操作。
实施例1
一种阴离子多肽羧基化生物纳米磁珠，由嫁接有聚乙二醇的阴离子多肽聚合物通过柔性linker连接到生物纳米磁珠的膜蛋白上融合表达而成，所述柔性linker的氨基酸残基为GCVA(DLGGV)2GVC(GA)3MADEGAG；所述阴离子多肽聚合物的氨基酸残基为AQEKNEEEETATEETEEEGAEGAEAEEEEETAEGAEDEDEDEEDGSGSQEHEEDEETEETEEGAEGEAEEAEDEAEEEDPGEEEDAQEEEEEGSGSEEEEEAQEEEDE。
实施例2
一种阴离子多肽羧基化生物纳米磁珠，由嫁接有聚乙二醇的阴离子多肽聚合物通过柔性linker连接到生物纳米磁珠的膜蛋白上融合表达而成，所述柔性linker的氨基酸残基为GCVA(DLGGV)2GVC(GA)3MADEGAG；所述阴离子多肽聚合物的氨基酸残基为GEFDDDDDDDDDFDEEFDDDDDDDDDGDDKDDDLDGDDDDDNDGSDEGSDDEDDDDDDDGDDEHHDDDGDDDPDHDDDHDDNNDDHDDDDNDHHDTDDPDHDDDHDDDDDDNNDDDNDDDDD。
实施例3
一种阴离子多肽羧基化生物纳米磁珠，由嫁接有聚乙二醇的阴离子多肽聚合物通过柔性linker连接到生物纳米磁珠的膜蛋白上融合表达而成，所述柔性linker的氨基酸残基为GCVA(DLGGV)2GVC(GA)3MADEGAG；所述阴离子多肽聚合物的氨基酸残基为AQEKNEEEETATEETEEEGAEGAEAEEEEETAEGAEDEDEDEEDGSGSQEHEEDEETEETEEGAEGEAEEAEDEAEEEDPGEEEDAQEEEEEGSGSEEEEEAQEEEDE。
制备方法：
A、构建细菌磁颗粒膜蛋白基因mamC或mamF缺失的突变体菌株，得到一级重组菌株；
B、通过DNA合成的方法制备阴离子多肽，将阴离子多肽与细菌磁颗粒膜蛋白基因mamC或mamF通过柔性linker进行融合形成基因融合表达载体；
C、将步骤B得到的表达载体导入到步骤A得到的一级重组菌株中，筛选出表达阴离子多肽的二级重组菌株；
D、将步骤C得到的二级重组菌种进行发酵培养，生产表达展示阴离子多肽的改性生物纳米磁珠；
E、采用多聚物糖基化聚乙二醇PEG对步骤D所得到的改性生物纳米磁珠进行嫁接修饰形成具有壳结构的所述阴离子多肽羧基化生物纳米磁珠。
其中，制备膜蛋白基因mamC或mamF缺失的突变体菌株的方法，基因融合表达载体的制备方法，表达载体的导入、菌株的筛选、菌种的发酵培养和从菌种中得到生物纳米磁珠的方法均可采用现有技术来实现，本实施例不做具体限定。
实施例4
一种制备如实施例1所提供的阴离子多肽羧基化生物纳米磁珠的方法，所述菌株选择MSR-Ⅰ野生型菌株，包括如下步骤：
（a-1）基因敲除：扩增细菌磁颗粒膜蛋白mamC基因左右两侧共两个长均为300bp的同源DNA片段，将所述DNA片段克隆在噬菌体病毒AAV-del微载体上，即得一条基于噬菌体病毒的微载体序列AAV-del-mac；
（a-2）基因转移：获取AAV-del-mac的核酸序列产物，调节核酸序列产物的浓度为1mg/ml，通过电转化的方式转入MSR-Ⅰ野生菌株中；采用方波电脉冲，电压为3100V，电脉冲时间是3.1ms，电脉冲次数是2次；
（a-3）菌株筛选：经过梯度筛选，获得mamc缺失突变的重组菌株，验证后即得一级重组菌株MSRⅠ-dC；
B、通过DNA合成的方法制备阴离子多肽，将阴离子多肽与细菌磁颗粒膜蛋白基因mamC通过柔性linker进行融合形成基因融合表达载体；
C、将步骤B得到的表达载体导入到步骤A得到的一级重组菌株MSRⅠ-dC中，筛选出表达阴离子多肽的二级重组菌株；
D、将步骤C得到的二级重组菌种进行发酵培养，分离纯化生产表达展示阴离子多肽的改性生物纳米磁珠；
E、采用多聚物糖基化聚乙二醇PEG对步骤D所得到的改性生物纳米磁珠进行嫁接修饰形成具有壳结构的所述阴离子多肽羧基化生物纳米磁珠。
实施例5
一种制备如实施例1和实施例2所提供的阴离子多肽羧基化生物纳米磁珠的方法，所述菌株选择MSR-Ⅰ野生型菌株，包括如下步骤：
（a-1）基因敲除：扩增细菌磁颗粒膜蛋白mamF基因左右两侧共两个长均为700bp的同源DNA片段，将所述DNA片段克隆在噬菌体病毒AAV-del微载体上，即得一条基于噬菌体病毒的微载体序列AAV-del-maf；
（a-2）基因转移：通过质粒提取及酶切方法获取AAV-del-maf的核酸序列产物，调节核酸序列产物的浓度为3mg/ml，通过电转化的方式转入MSR-Ⅰ野生菌株中；采用方波电脉冲，电压为3200V，电脉冲时间是3.3ms，电脉冲次数是1次；
（a-3）菌株筛选：经过梯度筛选，获得mamf缺失突变的重组菌株，验证后即得一级重组菌株MSRⅠ-dF；
B、通过DNA合成的方法制备阴离子多肽YR-APE1和YR-APD2的表达基因序列，将阴离子多肽YR-APE1和YR-APD2与细菌磁颗粒膜蛋白基因mamF通过柔性linker进行融合形成基因融合表达载体pmamF-APE1和pmamF-APD2；
C、将pmamF-APE1和pmamF-APD2，克隆到表达载体pBRC上，分别得到两个表达质粒pBRC-pmamF-APE1，pBRC-pmamF-APD2两个新的融合基因片段，通过三亲本接合的方式分别将pBRC-pmamF-APE1，pBRC-pmamF-APD2，分别转入一级重组菌MSRⅠ-dF中，验证正确后得到表达不同阴离子多肽的重组菌株，即二级重组菌株，分别命名为：MSRⅠ-dF/APE1，MSRⅠ-dF/APD2；
D、将步骤C得到的二级重组菌种进行发酵培养，分离纯化生产表达展示阴离子多肽的改性生物纳米磁珠；
E、采用多聚物糖基化聚乙二醇PEG对步骤D所得到的改性生物纳米磁珠进行嫁接修饰形成具有壳结构的所述阴离子多肽羧基化生物纳米磁珠。
实施例6
一种制备阴离子多肽羧基化生物纳米磁珠的方法，包括如下步骤：
（a-1）基因敲除：扩增细菌磁颗粒膜蛋白mamC基因左右两侧共两个长均为500bp的同源DNA片段，将所述DNA片段克隆在噬菌体病毒AAV-del微载体上，即得一条基于噬菌体病毒的微载体序列AAV-del-mac；
（a-2）基因转移：获取AAV-del-mac的核酸序列产物，调节核酸序列产物的浓度为2mg/ml，通过电转化的方式转入MSR-Ⅰ野生菌株中；采用方波电脉冲，电压为3100V，电脉冲时间是3.2ms，电脉冲次数是2次；
（a-3）菌株筛选：电转化后菌株通过蔗糖和庆大霉素梯度浓度压力筛选，获得mamc缺失突变的重组菌株，经过测序验证后即得一级重组菌株MSRⅠ-dC；
B、通过DNA合成的方法制备阴离子多肽YR-APE1的表达基因序列，将阴离子多肽YR-APE1与细菌磁颗粒膜蛋白基因mamC通过柔性linker进行融合形成基因融合表达载体pmamC-APE1；
C、将pmamC-APE1克隆到表达载体pBRC上，得到两个表达质粒pBRC-pmamC-APE1新的融合基因片段，通过电转化的方式将pBRC-pmamF- APE1转入一级重组菌MSRⅠ-dC中，验证正确后得到表阴离子多肽的重组菌株，即二级重组菌株命名为：MSRⅠ-dC/APE1；
d-1、预培养：将步骤C得到的二级重组菌株接种到经过灭菌后的第一培养基中培养14h后得到预培养菌株，预培养条件为：温度35℃，通气量为每分钟每1mL培养基通入0.5mL的气体，所述气体为5%O2 和95%N2的混合气体；
d-2、将所得的预培养菌株接种至装有灭菌第二培养基的发酵罐中进行深层培养3天后得到深层培养物，所述深层培养条件为：温度37℃，通气量为每分钟每1mL培养基通入0.4mL的气体，所述气体为5%O2、1%H2和94%N2的混合气体；
d-3、将得到的深层培养物依次进行菌体粉碎、磁力吸附和梯度纯化步骤，最终得到所述表达展示阴离子多肽的改性生物纳米磁珠；
E、采用多聚物糖基化聚乙二醇PEG对步骤d-3所得到的改性生物纳米磁珠进行嫁接修饰形成具有壳结构的所述阴离子多肽羧基化生物纳米磁珠。
实施例7
一种制备阴离子多肽羧基化生物纳米磁珠的方法，与实施例4的区别在于，步骤D包括如下具体步骤：
d-1、预培养：将步骤C得到的二级重组菌株接种到经过灭菌后的第一培养基中培养18h后得到预培养菌株，预培养条件为：温度37℃，通气量为每分钟每1mL培养基通入0.3mL的气体，所述气体为10%O2和90%N2的混合气体；
d-2、将所得的预培养菌株接种至装有灭菌第二培养基的发酵罐中进行深层培养4天后得到深层培养物，所述深层培养条件为：温度37℃，通气量为每分钟每1mL培养基通入0.6mL的气体，所述气体为5%O2、1%H2和94%N2的混合气体；
d-3、将得到的深层培养物依次进行菌体粉碎、磁力吸附和梯度纯化步骤，最终得到所述表达展示阴离子多肽的改性生物纳米磁珠。
实施例8
一种制备阴离子多肽羧基化生物纳米磁珠的方法，与实施例4的区别在于，步骤D包括如下具体步骤：
d-1、预培养：将步骤C得到的二级重组菌株接种到经过灭菌后的第一培养基中培养16h后得到预培养菌株，预培养条件为：温度38℃，通气量为每分钟每1mL培养基通入0.4mL的气体，所述气体为7%O2和93%N2的混合气体
d-2、将所得的预培养菌株接种至装有灭菌第二培养基的发酵罐中进行深层培养4天后得到深层培养物，所述深层培养条件为：温度34℃，通气量为每分钟每1mL培养基通入0.5mL的气体，所述气体为5%O2、1%H2和94%N2的混合气体；
d-3、将得到的深层培养物依次进行菌体粉碎、磁力吸附和梯度纯化步骤，最终得到所述表达展示阴离子多肽的改性生物纳米磁珠。
所述第一培养基由重量份数为1份腐胺二盐酸盐、0.2份多氯化胆碱、8份D-葡萄糖、2份亚油酸、2份硫代甘油、1份乙酸钠、3琼脂和2份海藻酸铵组成；所述第二培养基由重量份数为3份右旋糖酐、2份吐温80、0.5份甲砜霉素、8份D-葡萄糖、2份亚油酸、3份海藻糖和1份硫代甘油组成。
实施例9
一种制备阴离子多肽羧基化生物纳米磁珠的方法，与实施例8的区别在于，所述第一培养基由重量份数为2份腐胺二盐酸盐、0.1份多氯化胆碱、7份D-葡萄糖、1份亚油酸、3份硫代甘油、0.5份乙酸钠、5琼脂和1份海藻酸铵组成；所述第二培养基由重量份数为2份右旋糖酐、3份吐温80、0.3份甲砜霉素、7份D-葡萄糖、1份亚油酸、2份海藻糖和2份硫代甘油组成。
实施例10
一种制备阴离子多肽羧基化生物纳米磁珠，与实施例4的区别在于，述步骤E包括如下具体步骤：
e-1、取20mg的生物纳米磁珠溶于10mL磷酸缓冲液中，优选浓度为0.2%，加入1.5mL浓度为9mM的N乙酰羧基糖，室温搅拌30min；
e-2、加入N乙酰羧基糖转移酶，至终浓度45mU，室温搅拌3h，得到UDP-GalNAc定点修饰的生物纳米磁珠；
e-3、将步骤e-2得到的生物纳米磁珠采用磁力架分离纯化，洗涤后，重新溶于10mL磷酸缓冲液中，加入1mL浓度为2.5mM的唾液酸活化PEG，搅拌混匀；然后加入唾液酸转移酶，至终浓度200mU，于32℃，以80rpm的速度，轻轻摇晃反应24小时，催化形成具有PEG聚合物外壳的生物纳米磁珠；
e-4、将步骤e-3得到的生物纳米磁珠用25%乙醇洗涤2次，即得所述具有壳结构的阴离子多肽羧基化生物纳米磁珠。
实施例11
一种制备阴离子多肽羧基化生物纳米磁珠，与实施例7的区别在于，述步骤E包括如下具体步骤：
e-1、取30mg的生物纳米磁珠溶于12mL磷酸缓冲液中，优选浓度为0.3%，加入1mL浓度为9mM的N乙酰羧基糖，室温搅拌30min；
e-2、加入N乙酰羧基糖转移酶，至终浓度35mU，室温搅拌5h，得到UDP-GalNAc定点修饰的生物纳米磁珠；
e-3、将步骤e-2得到的生物纳米磁珠采用磁力架分离纯化，洗涤后，重新溶于12mL磷酸缓冲液中，加入2mL浓度为2.5mM的唾液酸活化PEG，搅拌混匀；然后加入唾液酸转移酶，至终浓度280mU，于32℃，以50rpm的速度，轻轻摇晃反应48小时，催化形成具有PEG聚合物外壳的生物纳米磁珠；
e-4、将步骤e-3得到的生物纳米磁珠用25%乙醇洗涤3次，即得所述具有壳结构的阴离子多肽羧基化生物纳米磁珠。
实施例12
一种制备阴离子多肽羧基化生物纳米磁珠，与实施例8的区别在于，述步骤E包括如下具体步骤：
e-1、取25mg的生物纳米磁珠溶于10mL磷酸缓冲液中，优选浓度为0.3%，加入1mL浓度为9mM的N乙酰羧基糖，室温搅拌30min；
e-2、加入N乙酰羧基糖转移酶，至终浓度40mU，室温搅拌4h，得到UDP-GalNAc定点修饰的生物纳米磁珠；
e-3、将步骤e-2得到的生物纳米磁珠采用磁力架分离纯化，洗涤后，重新溶于10mL磷酸缓冲液中，加入1mL浓度为2.5mM的唾液酸活化PEG，搅拌混匀；然后加入唾液酸转移酶，至终浓度250mU，于32℃，以70rpm的速度，轻轻摇晃反应36小时，催化形成具有PEG聚合物外壳的生物纳米磁珠；
e-4、将步骤e-3得到的生物纳米磁珠用25%乙醇洗涤3次，即得所述具有壳结构的阴离子多肽羧基化生物纳米磁珠。
对照例1
一种阴离子多肽羧基化生物纳米磁珠，由嫁接有聚乙二醇的阴离子多肽聚合物通过柔性linker连接到生物纳米磁珠的膜蛋白上融合表达而成，所述柔性linker的氨基酸残基为GCVADLGGVGVCGAMADEGAG；所述阴离子多肽聚合物的氨基酸残基为AQEKNEEEETATEETEEEGAEGAEAEEEEETAEGAEDEDEDEEDGSGSQEHEEDEETEETEEGAEGEAEEAEDEAEEEDPGEEEDAQEEEEEGSGSEEEEEAQEEEDE。
对照例2
一种阴离子多肽羧基化生物纳米磁珠，由嫁接有聚乙二醇的阴离子多肽聚合物通过柔性linker连接到生物纳米磁珠的膜蛋白上融合表达而成，所述柔性linker的氨基酸残基为GCVADLGAGAMGVGVCGGVDLGGAADEGAG；所述阴离子多肽聚合物的氨基酸残基为AQEKNEEEETATEETEEEGAEGAEAEEEEETAEGAEDEDEDEEDGSGSQEHEEDEETEETEEGAEGEAEEAEDEAEEEDPGEEEDAQEEEEEGSGSEEEEEAQEEEDE。
对照例3
一种阴离子多肽羧基化生物纳米磁珠，由嫁接有聚乙二醇的阴离子多肽聚合物通过柔性linker连接到生物纳米磁珠的膜蛋白上融合表达而成，所述柔性linker的氨基酸残基为GCVA(DLGGV)2GVC(GA)3M(ADEGAG)2；所述阴离子多肽聚合物的氨基酸残基为AQEKNEEEETATEETEEEGAEGAEAEEEEETAEGAEDEDEDEEDGSGSQEHEEDEETEETEEGAEGEAEEAEDEAEEEDPGEEEDAQEEEEEGSGSEEEEEAQEEEDE。
对照例4
一种阴离子多肽羧基化生物纳米磁珠，由嫁接有聚乙二醇的阴离子多肽聚合物通过柔性linker连接到生物纳米磁珠的膜蛋白上融合表达而成，所述柔性linker的氨基酸残基为GCVA(DLGGV)2GVC(GA)3M(ADEGAG)2；所述阴离子多肽聚合物的氨基酸残基为AKNEEQEEETATEETEEEGAEAEEEGAEEEDEEDGSGSQETADEEGAEDEDEETHEEEETEEGAEGEAEEGEEEDAQEEEAEDEAEEEDPEEEGSGSEEEEAEEQEDE。
对照例5
一种制备如实施例1所提供的阴离子多肽羧基化生物纳米磁珠的方法，所述菌株选择MSR-Ⅰ野生型菌株，与实施例4的区别在于，步骤（a-2）获取AAV-del-mac的核酸序列产物，通过亲本结合的方式转入MSR-Ⅰ野生菌株中。
对照例6-11
制备如实施例1所提供的阴离子多肽羧基化生物纳米磁珠的方法，与实施例4的区别在于，所述电转化的具体参数如表1。
表1各对照例的电转化具体参数
组别 电压V 电脉冲时间ms 电脉冲次数
对照例6 2900V 3.1ms 2
对照例7 3000V 3.1ms 2
对照例8 3300V 3.1ms 2
对照例9 3100V 3.0ms 2
对照例10 3100V 3.4ms 2
对照例11 3100V 3.1ms 3
对照例12-16
制备如实施例1所提供的阴离子多肽羧基化生物纳米磁珠的方法，与实施例7的区别在于，所述培养的具体参数如表2。
表2各对照例的发酵培养具体参数
组别 预培养 深层培养
温度 通气量 含氧量 温度 通气量 含氧和氢的量
对照例12 37℃ 0.3mL 12%O2 37℃ 0.6mL 5%O2
对照例13 37℃ 0.3mL 18%O2 37℃ 0.6mL 5%O2+3%H2
对照例14 37℃ 0.3mL 3%O2 37℃ 0.6mL 10%O2+1%H2
对照例15 25℃ 0.3mL 10%O2 28℃ 0.6mL 5%O2+1%H2
对照例16 37℃ 0.2mL 10%O2 37℃ 1mL 5%O2+1%H2
试验例1阴离子多肽羧基化生物纳米磁珠的生物纳米磁珠载量试验
1.1试验分组
取实施例1-3和对照例1-11所提供的生物纳米磁珠（任意相同的常规方法制备而成）以及实施例4的制备方法所得到的生物纳米磁珠进行试验。
1.2试验方法
将每组的生物纳米磁珠通过磁力吸附后吸干水分称量重量M0，加入保存液重悬磁珠，使磁珠的浓度为1mg/mL。
购买FITC-Ab-EDC抗体，调节好浓度为1mg/mL，按照1/10的梯度系列稀释，通过荧光分析仪计算每个梯度的荧光强度，制作荧光强度标准曲线；
另取0.5mL浓度为0.1mg/mL的FITC-Ab-EDC抗体，加入100μL纳米磁珠，混匀，37℃温育15min，其间混匀3-5次；用磁力吸附磁珠，吸取上清，检测上清的FITC荧光强度，同时对磁珠进行数次洗涤，去掉结合不牢固的抗体，重悬磁珠后检测溶液中的荧光强度；
最后通过标准曲线可计算荧光强度对应的抗体数量，通过间接和直接两种方法，测量纳米磁珠实际标记上抗体的载量，结果见表3，其中结合抗体量为1mg生物纳米磁珠结合的抗体量。
表3各组1mg生物纳米磁珠的抗体结合量
组别 结合抗体量（μg） 组别 结合抗体量（μg） 组别 结合抗体量（μg）
实施例1 127 对照例2 75 对照例7 55
实施例2 123 对照例3 83 对照例8 73
实施例3 155 对照例4 81 对照例9 84
实施例4 179 对照例5 70 对照例10 50
对照例1 72 对照例6 62 对照例11 63
1.3结果
由上述试验结果可知，本发明所提供的阴离子多肽羧基化生物纳米磁珠，阴离子多肽通过特定适宜的linker连接在纳米磁珠的膜蛋白上，三者相互结合性好，产生的可结合羧基基位点数量多，结合抗体量高，实用性好；其中通过实施例4提供的方法制备的生物纳米磁珠，结合抗体量最高，性能最好。对照例1-4所提供的生物纳米磁珠，对柔性Linker和结合的蛋白进行考察，结果可知linker的微小改变对得到的生物纳米磁珠的载量具有较大影响；对照例6-11提供的方法制备的生物纳米磁珠可知，本发明所限定的制备方法具有突出的效果，可以有效提高产品的载量和性能。
试验例2阴离子多肽羧基化生物纳米磁珠的稳定性试验（高温加速试验）
2.1试验方法
取实施例1-4和对照例1-11经过试验例1得到的FITC-Ab-EDC抗体偶联的生物纳米磁珠，每组均分为14支，共同至于37℃条件下，每天每组分别取出1支，用PBS洗涤数次，磁力吸附后重悬，检测其FITC荧光强度，每次均与正常4℃保存的试剂荧光强度进行比较，最后得到纳米磁珠试剂荧光强度的衰减曲线，以衰减>35%为企业内部标准，设为试剂失效的保存时间，结果见表4。
表4各组生物纳米磁珠的高速加速试验结果
组别 失效时间（天） 组别 失效时间（天） 组别 失效时间（天）
实施例1 14 对照例2 7 对照例7 11
实施例2 14 对照例3 7 对照例8 12
实施例3 -- 对照例4 8 对照例9 12
实施例4 -- 对照例5 6 对照例10 10
对照例1 8 对照例6 12 对照例11 11
注：“--”表明第14天时依旧保持70%以上的活性。
2.2结果
通常37摄氏度放置1天约为4℃保存40天，试剂正常保存有效期使用期限为1年。由上述试验结果可知，本发明所提供的生物纳米磁珠一般在14天依旧能保持原有活性的60%左右，而通过实施例3和实施例4的制备方法得到的生物纳米磁珠，在14天后依旧保持原有活性的75%左右，而对照例提供的生物纳米磁珠稳定性较差；本发明提供的生物纳米磁珠稳定性高，对环境的耐受性好，具有较好的经济效益。
试验例3阴离子多肽羧基化生物纳米磁珠产量及菌株活性试验
3.1试验分组
取实施例4、7、8和对照例5-16的制备方法所得到的生物纳米磁珠进行试验。
3.2试验方法
在实施例4、7、8和对照例5-16的（a-2）步骤中，每次电转化107个细菌数目，转化1μg量的DNA（大小约5kbp）作为标准实验，检测电转化完毕的细菌成活率（%），在步骤（a-3）之后，检测DNA转化表达成功率（%），取十分之一进行稀释涂平板，检测到每块单克隆菌落数目>300个；另外在步骤D（d-3）结束后，检测每升发酵液所得到的改性生物纳米磁珠量，结果见表5。
表5各组生物纳米磁珠的活性及产量
组别 细菌成活率（%） 转化成功率（%） 磁珠量（mg） 组别 细菌成活率（%） 转化成功率（%） 磁珠量(mg)
实施例4 87 62 208 对照例10 37 43 37
实施例7 85 69 257 对照例11 29 39 35
实施例8 89 65 289 对照例12 83 61 152
对照例5 80 12 57 对照例13 82 62 177
对照例6 84 31 134 对照例14 86 61 163
对照例7 78 35 127 对照例15 85 60 169
对照例8 32 46 43 对照例16 87 60 149
对照例9 66 41 55
3.3结果
由上述试验结果可知，通过本发明提供的制备方法制备过程中菌株成活率高，同时DNA转化表达成功率高，得到的生物纳米磁珠量大，与对照组具有显著性差异；其中，实施例7得到的生物纳米磁珠产量较实施例4高，实施例8的产量最高；对照例5-11是以实施例1为基础，对基因转移的方式进行考察，可以看出对照例5的磁珠活率较高，但是DNA转化表达率成功率很低，导致得到的磁珠数量也较少；对照例6-11的电转化条件的改变，对MSR-Ⅰ菌株的适应性较差，转化成功率和菌株成活率不能同时达到最优；而对照例12-16与实施例7相比较，发现发酵培养过程中的气体通入量与通入的成分，对于菌株的成活，纳米磁珠的产量具有较大影响，本发明通过对发酵过程中通入气体进行改进，在提高纳米磁珠产量上具有突出的实际效果。

Claims (9)

  1. 一种阴离子多肽羧基化生物纳米磁珠，其特征在于，所述阴离子多肽羧基化生物纳米磁珠由嫁接有聚乙二醇的阴离子多肽聚合物通过柔性linker连接到生物纳米磁珠的膜蛋白上融合表达而成，所述柔性linker的氨基酸残基为GCVA(DLGGV)2GVC(GA)3MADEGAG。
  2. 如权利要求1所述的阴离子多肽羧基化生物纳米磁珠，其特征在于，所述阴离子多肽聚合物的氨基酸残基为AQEKNEEEETATEETEEEGAEGAEAEEEEETAEGAEDEDEDEEDGSGSQEHEEDEETEETEEGAEGEAEEAEDEAEEEDPGEEEDAQEEEEEGSGSEEEEEAQEEEDE。
  3. 如权利要求1所述的阴离子多肽羧基化生物纳米磁珠，其特征在于，所述阴离子多肽聚合物的氨基酸残基为GEFDDDDDDDDDFDEEFDDDDDDDDDGDDKDDDLDGDDDDDNDGSDEGSDDEDDDDDDDGDDEHHDDDGDDDPDHDDDHDDNNDDHDDDDNDHHDTDDPDHDDDHDDDDDDNNDDDNDDDDD。
  4. 一种权利要求1所述的阴离子多肽羧基化生物纳米磁珠的制备方法，其特征在于，所述方法包括如下步骤：
    A、构建细菌磁颗粒膜蛋白基因mamC或mamF缺失的突变体菌株，得到一级重组菌株；
    B、通过DNA合成的方法制备阴离子多肽，将阴离子多肽与细菌磁颗粒膜蛋白基因mamC或mamF通过柔性linker进行融合形成基因融合表达载体；
    C、将步骤B得到的表达载体导入到步骤A得到的一级重组菌株中，筛选出表达阴离子多肽的二级重组菌株；
    D、将步骤C得到的二级重组菌种进行发酵培养，分离纯化生产表达展示阴离子多肽的改性生物纳米磁珠；
    E、采用多聚物糖基化聚乙二醇对步骤D所得到的改性生物纳米磁珠进行嫁接修饰形成具有壳结构的所述阴离子多肽羧基化生物纳米磁珠。
  5. 如权利要求4所述的制备方法，其特征在于，所述菌株选择MSR-Ⅰ野生型菌株，所述步骤A的具体方法包括如下步骤：
    （a-1）基因敲除：扩增细菌磁颗粒膜蛋白mamC或mamF基因左右两侧共两个长均为300-700bp的同源DNA片段，将所述DNA片段克隆在噬菌体病毒AAV-del微载体上，即得一条基于噬菌体病毒的微载体序列AAV-del-mac或AAV-del-maf；
    （a-2）基因转移：获取AAV-del-mac或AAV-del-maf的核酸序列产物，调节核酸序列产物的浓度为1-3mg/ml，通过电转化的方式转入MSR-Ⅰ野生菌株中；
    （a-3）菌株筛选：经过梯度筛选，获得mamc或mamf缺失突变的重组菌株，验证后即得一级重组菌株MSRⅠ-dC或MSRⅠ-dF。
  6. 如权利要求5所述的制备方法，其特征在于，所述电转化的具体方案为：采用方波电脉冲，电压为3100V-3200V，电脉冲时间是3.1-3.3ms，电脉冲次数是1-2次。
  7. 如权利要求4所述的制备方法，其特征在于，所述步骤D包括如下具体步骤：
    d-1、预培养：将步骤C得到的二级重组菌株接种到经过灭菌后的第一培养基中培养14-18h后得到预培养菌株，预培养条件为：温度35-38℃，通气量为每分钟每1mL培养基通入0.3-0.5mL的气体，所述气体为5%-10%O2和90%-95%N2的混合气体；
    d-2、将所得的预培养菌株接种至装有灭菌第二培养基的发酵罐中进行深层培养3-4天后得到深层培养物，所述深层培养条件为：温度34-37℃，通气量为每分钟每1mL培养基通入0.4-0.6mL的气体，所述气体为5%O2、1%H2和94%N2的混合气体；
    d-3、将得到的深层培养物依次进行菌体粉碎、磁力吸附和梯度纯化步骤，最终得到所述表达展示阴离子多肽的改性生物纳米磁珠。
  8. 如权利要求7所述的制备方法，其特征在于，所述第一培养基由重量份数为1-2份腐胺二盐酸盐、0.1-0.2份多氯化胆碱、7-8份D-葡萄糖、1-2份亚油酸、2-3份硫代甘油、0.5-1份乙酸钠、3-5琼脂和1-2份海藻酸铵组成；所述第二培养基由重量份数为2-3份右旋糖酐、2-3份吐温80、0.3-0.5份甲砜霉素、7-8份D-葡萄糖、1-2份亚油酸、2-3份海藻糖和1-2份硫代甘油组成。
  9. 如权利要求4所述的制备方法，其特征在于，所述步骤E由如下分步骤构成：
    e-1、取20-30mg的生物纳米磁珠溶于10-12mL磷酸缓冲液中，优选浓度为0.2-0.3%，加入1-1.5mL浓度为9mM的N乙酰羧基糖，室温搅拌30min；
    e-2、加入N乙酰羧基糖转移酶，至终浓度35-45mU，室温搅拌3-5h，得到UDP-GalNAc定点修饰的生物纳米磁珠；
    e-3、将步骤e-2得到的生物纳米磁珠采用磁力架分离纯化，洗涤后，重新溶于10-12mL磷酸缓冲液中，加入1-2mL浓度为2.5mM的唾液酸活化PEG，搅拌混匀；然后加入唾液酸转移酶，至终浓度200-280mU，于32℃，以50-80rpm的速度，轻轻摇晃反应24-48小时，催化形成具有PEG聚合物外壳的生物纳米磁珠；
    e-4、将步骤e-3得到的生物纳米磁珠用25%乙醇洗涤2-3次，即得所述具有壳结构的阴离子多肽羧基化生物纳米磁珠。
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