WO2007140667A1 - Protéine de fusion ptd-vp3 utilisée comme médicament anti-tumoral et procédé de préparation - Google Patents

Protéine de fusion ptd-vp3 utilisée comme médicament anti-tumoral et procédé de préparation Download PDF

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WO2007140667A1
WO2007140667A1 PCT/CN2006/003636 CN2006003636W WO2007140667A1 WO 2007140667 A1 WO2007140667 A1 WO 2007140667A1 CN 2006003636 W CN2006003636 W CN 2006003636W WO 2007140667 A1 WO2007140667 A1 WO 2007140667A1
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ptd4
protein
fusion protein
transduction domain
chicken anemia
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PCT/CN2006/003636
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English (en)
Chinese (zh)
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Shen Qu
Jun Sun
Yiqiang Zong
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Huazhong University Of Science And Technology
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/10011—Circoviridae
    • C12N2750/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present patent application relates to biotechnology, and more particularly to biotechnological drugs, and more particularly to biotechnological drugs for treating tumors.
  • Tumors are the main diseases causing death in the world.
  • the treatment methods for tumors include surgical treatment, radiotherapy and chemotherapy.
  • Surgical treatment has only a certain effect on early tumors, and is not effective for advanced tumors, radiotherapy and chemotherapy. High cost and great damage to human body. Finding new safe and effective drugs and methods for treating tumors is an important issue in the current medical field.
  • the VP3 protein also known as apoptin
  • CAV chicken anemia virus
  • VP3 (referred to as VP3 protein of chicken anemia virus, CAV VP3) can induce apoptosis of various tumor cells, and its induced apoptosis effect is non-p53-dependent [2] Not inhibited by anti-apoptotic factors Bel-2, Bel-xL [3] . More significantly, VP3 only induced apoptosis with tumorigenic phenotypic cells or transformed phenotype cells, whereas TP3 transgenic mice with normal apoptotic and non-cytotoxic effects on TH cells were able to grow normally [51 , these characteristics It indicates that VP3 is a promising anti-tumor preparation.
  • Protein transduction technology is a new macromolecular transfer technology in recent years, which utilizes some protei n transducting domain (PTD) (Chinese abbreviation: protein transduction domain). Proteins or peptides directly "transfer" therapeutic macromolecules into cells to exert biological effects [1Q] .
  • PTD protei n transducting domain
  • CPPs Cell l-Penetrati ng Peptides
  • ⁇ 11 peptide, 13 peptide
  • Antp 16 peptide
  • VP22 34 peptide
  • Transportan 28 peptide
  • MAP MAP
  • CPP can transport a wide range of substances, such as proteins, DNA, antibodies, imaging agents, toxins, nano drug particles, liposomes, etc. [1 ' ] .
  • the CPP delivery system is both a good tool for studying intracellular biological processes and a tool for biopharmaceutical delivery.
  • TAT trans-activator of transcription
  • the TAT protein (86 peptide) is a transcriptional activator protein.
  • Green [ 11] and Frankel [12] independently found that HIV-TAT protein has the ability to penetrate biofilm [ 12-13] ; The ability is mediated by a peptide between 47-57 (or 48-60) amino acid residues, and the fusion protein formed by the TAT polypeptide with other proteins can also penetrate the cell membrane and exert the biological functions of these proteins [ 14 _ 16] .
  • Ho et al. [17] used a chemical synthesis method to transform TAT, and obtained a series of TAT-PTD peptides by amino acid substitution.
  • the secondary structure of PTD4 is more stable and the transduction efficiency is higher, which can successfully transduce the target protein.
  • the rate is almost 100%, and the amount of protein introduced by a single cell is 33 times that of TAT.
  • whether the series of PTD can realize transmembrane transport of fusion protein through biosynthesis has not been reported yet. Summary of the invention
  • the object of the present invention is to provide a tumor transduction domain-chicken anemia virus protein 3 fusion protein (PTD-VP3 fusion protein, or PTD-CAV VP3 fusion protein), which has a good penetration biofilm effect. Moreover, it can induce tumor cell apoptosis, has greater lethality to tumor cells, does not damage normal cells, and does not have the introduction of foreign genes, especially the fusion protein drug can be applied by skin application. medicine. At the same time, the present invention also provides a method for preparing such a tumor-treating fusion protein.
  • PTD-VP3 fusion protein or PTD-CAV VP3 fusion protein
  • the technical scheme for realizing the present invention is: preparing a protein transduction domain-chicken anemia virus protein 3 fusion protein PTD-VP3, and directly introducing the VP3 protein into the cell by using the protein transduction domain PTD, and then specifically inducing apoptosis of the tumor cell by using VP3. Characteristics, thereby achieving the purpose of promoting tumor apoptosis and inhibiting tumor growth.
  • the PTD4-VP3 fusion protein was prepared, and the VP3 protein was directly introduced into the cell by PTD4, and then the VP3 was specifically used to induce the apoptosis of the tumor cell, thereby promoting tumor apoptosis and inhibiting tumor growth. purpose.
  • the protein transduction domain 4-chicken anemia virus protein 3 fusion protein provided by the invention has the amino acid sequence shown in sequence 6 or sequence 7 in the sequence listing; the expressed protein transduction domain 4-chicken anemia virus protein 3 fusion protein provided by the invention
  • the gene has the nucleotide sequence shown in SEQ ID NO: 5 in the sequence listing;
  • the method for preparing the protein transduction domain 4-chicken anemia virus protein 3 fusion protein provided by the present invention comprises the following steps: a. constructing prokaryotic expression containing the PTD4 sequence Vector pET28a- PTD4;
  • the method for constructing the prokaryotic expression vector pET28a-PTD4 containing the PTD4 sequence as described in a. above is:
  • the 11 amino acid sequence of the PTD4 polypeptide is designed according to the characteristics of the prokaryotic codon, and the two oligonucleotide base sequences encoding the PTD4 polypeptide are designed.
  • the two oligonucleotide base sequences encoding the PTD4 polypeptide have the sequence 2 in the sequence listing.
  • the nucleotide sequence shown, the two oligonucleotide fragments were synthesized, and the two oligonucleotide fragments were mixed in an equimolar manner at 95 ° C for 10 min, then left at room temperature for 1 h to form double-stranded DNA encoding PTD4.
  • the obtained DNA double strand encoding PTD4 was inserted into Nhe I of the prokaryotic expression vector pET28a.
  • the method for constructing the prokaryotic expression vector pET28a-PTD4-VP3 containing the PTD4-VP3 gene described in the above b. is: constructing the eukaryotic expression vector pcDNA-VP3 containing the VP3 gene, and having the restriction endonuclease at the 5' end
  • the two PCR primers of EcoR I and Xho I recognition sites 5'-AGGAATTCATGAACGCTCTCCAAG-3' and 5'-GCGTCGACTTACAGTCTTATACGCC-3' were subjected to PCR amplification reaction to amplify the VP3 gene of chicken anemia virus under the reaction condition of 94.
  • the purified recombinant plasmid pET28a-PTD4-VP3 was transformed into the expression strain Escherichia coli BL21 (DE3) PlysS in 5 ml, containing 0.05 mg/ml, and the purified recombinant plasmid pET28a-PTD4-VP3 was expressed in the above.
  • the purified recombinant plasmid pET28a-PTD4-VP3 was expressed in the above.
  • OmM was induced for 8 hours, sonicated, and the supernatant was collected by centrifugation to induce bacteria
  • a control identified by 12.5% SDS-PAGE
  • the supernatant was dissolved in the loading buffer (using the loading buffer indicated in the operating manual provided by the company using the purchased nickel affinity chromatography column)
  • the nickel affinity chromatography column was purified, and the eluted protein was identified by SDS-PAGE.
  • the eluate containing the target protein was combined, dialyzed, concentrated, filtered and sterilized, and the protein content was determined by BCA method, and stored at -80 °C.
  • the PTD4 synthesized by the present invention has a function of mediating the penetration of the fusion protein into the biofilm. It was confirmed by TUNEL method and DAPI staining that the PTD4-VP3 fusion protein provided by the present invention can induce tumor cell apoptosis.
  • the PTD4-VP3 fusion protein provided by the present invention has a good penetrating biofilm effect and can induce tumor cell apoptosis, and has great killing ability to tumor cells.
  • the protein transduction domain 4-chicken anemia fusion protein 3 (PTD4-VP3) provided by the invention can be used for preparing an anti-tumor drug, for example, for preparing an anti-liver cancer drug, and the preparation method comprises the PTD4-VP3 fusion protein as an active ingredient, plus
  • the pharmaceutically acceptable carrier and/or additive is prepared into an antitumor pharmaceutical preparation in a conventional manner.
  • the carrier may be PBS (phosphate buffer), glycerol or urea or the like.
  • the fusion protein provided by the present invention is used as an active ingredient, and a carrier for preparing a transdermal preparation, such as phosphate buffer, glycerin, water, petrolatum, polyethylene glycol, etc., can be prepared into a transdermal preparation for treating tumors. .
  • a carrier for preparing a transdermal preparation such as phosphate buffer, glycerin, water, petrolatum, polyethylene glycol, etc.
  • Figure 1 is a schematic diagram showing the construction of a recombinant plasmid of the present invention
  • Figure 6 shows the results of fluorescence microscopy of the transmembrane effect of the PTD4-GFP fusion protein
  • Figure 7 shows the results of optical microscopy of the transmembrane effect of the PTD4-GFP fusion protein
  • PTD4-VP3 fusion protein induces apoptosis of HepG 2 cells by laser confocal microscopy to observe FITC excitation results
  • Figure 9 PTD4-VP3 fusion protein induces apoptosis of HepG 2 cells by laser confocal microscopy to observe DAPI excitation results
  • Figure 10 PTD4- The results of FITC and DAPI co-excitation were observed by laser confocal microscopy of VP3 fusion protein induced apoptosis of HepG 2 cells.
  • FIG 11 PTD4-VP3 fusion protein induced apoptosis of HepG2 cells by laser confocal microscopy;
  • Figure 12 Human cervical cancer Hela cell tumor-bearing nude mice tumor growth curve;
  • PTD4 polypeptide consists of 11 amino acids, and its base sequence is designed according to the characteristics of prokaryotic codons as follows:
  • the prokaryotic expression vector pET- 28a (+) was purchased from Novagen.
  • the double strand obtained by the above method was inserted into Nhe I of pET28a, and DH5 ⁇ was transformed, and the recombinant plasmid pET28a-PDD4 was constructed by restriction enzyme digestion, PCR and sequencing. DNA sequencing was performed by Shanghai Boya Company.
  • the primers for PCR identification are:
  • the cycle parameters were 95 ⁇ denaturation 5 min, 94 °C lmin, 50 °C lmin, 72 °C lmin, cycle amplification 30 times, and finally 72 °C extension 10 min e
  • Ml Mraker, molecular weight standard, from large to small, 7000, 5500, 3500, 2000, 1000, 500bp A: pET28a-PTD4 after Nhe I digestion
  • M2 Mraker, molecular weight standard, 600, 500, 400, 300, 200, lOObp from large to small. From the results of Figure 2, the recombinant pET28a-PTD4 was successfully constructed.
  • the 5' ends of the two primers contain recognition sites for the restriction enzymes EcoR I and Xho I Sal I, respectively.
  • PCR amplification reaction conditions The cycle parameters were 94 ° C for 5 min, 94 ° C for 55 sec, 60 ° C for 50 sec, 72 ° C for 55 sec, cycle amplification for 30 times, and incubation at 72 ° C for 10 min.
  • the PCR product was identified by 1.5% agarose gel electrophoresis.
  • the construction method uses a conventional gene cloning method.
  • PCR amplification fragment VP3 and pET28a- PTD4 recombinant plasmids were digested with EcoR I and Sal I, respectively, and the target fragment was recovered, ligated and transformed into DH5 a, and the plasmid was amplified.
  • Figure 3 shows the results of identification of recombinant pET28a- PTD4- VP3, in which:
  • Ml Marker, molecular weight standard, from 7000, 5500, 3500, 2000, 1000, 500bp
  • M3 raker, molecular weight standard, from large to small, 1500, 1000, 900, 800, 700, 600, 500, 400, 300, 200, lOObp
  • pEGFP-Cl was purchased from CLONETECH.
  • the 5' ends of the two primers contain recognition sites for the restriction enzymes BamH I and EcoR I, respectively.
  • the cycle parameters were 94 ° C for 5 min, 94 ° C for 55 sec, 62 ° C for 55 sec, 72 ° C for lmin, cycle amplification for 30 times, and finally for 72 min at 72 ° C.
  • the PCR product was identified by the 1.5% agarose gel electrophoresis.
  • step (1) The construction of prokaryotic expression vector pET28a PTD4 is shown in step (1).
  • Figure 4 shows the results of identification of recombinant pET28a- PTD4-GFP, in which:
  • Ml Marker, molecular weight standard, from 7000, 5500, 3500, 2000, 1000, 500bp
  • the purified two recombinant plasmids pET28a-PTD4-VP3 and pET28a-PTD4-GFP were transformed into the expression strain Escherichia coli BL2KDE3) PlysS, respectively, and shaken in 5 ml of LB medium containing 0.05 mg/ml kanamycin at 37 °C. The 5% of the SDS- was added to the A leg. The sputum was added to the final concentration. The OMP was induced for 8 hours, and the supernatant was removed by ultrasound. The supernatant was collected by centrifugation. PAGE identification.
  • the supernatant was dissolved in a loading buffer and purified by a nickel affinity chromatography column, and the operation was carried out according to the requirements of the kit.
  • the eluted protein was identified by SDS-PAGE, and the eluate containing the protein of interest was combined, dialyzed, concentrated, filtered and sterilized, and the protein content was determined by BCA method, - 8 (TC storage.
  • M is the molecular weight standard of the protein, from large to small: 116. 0 kDa, 66. 2 kDa, 45. 0 kDa, 35. 0 kDa, 25. 0 kDa, 18. 4kDa, 14. 4kDa.
  • PTD4-GFP in PBS phosphate buffer: 0. 8% NaCl, 0.02% KC1, 0. 144% ⁇ 3 ⁇ 4 ⁇ 0 formulate 0. 024% ⁇ 3 ⁇ 4 ⁇ 0 medicine.
  • the human hepatoma cell line pG 2 was inoculated into the culture plate. After the cells were attached, the cells were incubated with the mol/L fusion protein PTD4-GFP. After 2 hours, the culture solution was aspirated, washed three times with PBS, and then observed under a fluorescence microscope.
  • Fig. 6 is a fluorescence microscope for the membrane transmembrane effect of PTD4-GFP fusion protein. The results of the observation; Fig. 7 shows the results of optical microscopic observation of the transmembrane effect test of the PTD4-GFP fusion protein.
  • FIG. 7 of the result indicating success PTD4- GFP fusion protein through the cell membrane into the cells in G 2 H printed, synthesized PTD4 described the present invention provides a fusion protein having the ability to mediate permeable membrane.
  • PTD4-VP3 in PBS phosphate buffer: 0. 8% NaCl, 0.02% KC1, 0. 144% ⁇ 3 ⁇ 4 ⁇ 0 formulate 0. 024% ⁇ 2 ⁇ 0, ) solution.
  • the experimental cells were purchased from human hepatocarcinoma cell line ⁇ G 2 to purchase CCTCC.
  • the TUNEL test kit was purchased from Roche. 2.
  • the treated coverslips were placed in a six-well plate, human hepatoma cells HepG 2 were seeded in a six-well plate at a suitable density, and after ligation of the cells, lnol/L fusion proteins PTD4-VP3 and PTD4-GFP were used.
  • - VP3 was incubated for 4-5 days, washed three times with PBS, fixed with 4% paraformaldehyde for 30 min, apoptosis was detected by TUNEL (FITC staining) and counterstained with DAPI. The procedure was followed according to the instructions. Placed under a laser confocal microscope.
  • the PTD4-VP3 fusion protein provided by the present invention is used as an active ingredient, and PBS (phosphate buffer: 0.8% NaCl, 0.02% KC1, 0. 144% N3 ⁇ 4 HP0 4 , 0. 024% KH 2 P ( 3 ⁇ 4)
  • PBS phosphate buffer: 0.8% NaCl, 0.02% KC1, 0. 144% N3 ⁇ 4 HP0 4 , 0. 024% KH 2 P ( 3 ⁇ 4)
  • the solution is prepared into an antitumor drug preparation according to a conventional method.
  • the PTD4-VP3 fusion protein provided by the present invention is used as an active ingredient, and a PBS solution and 10% glycerol are added, and an antitumor drug preparation is prepared in a conventional manner.
  • the PTM-VP3 fusion protein provided by the present invention is used as an active ingredient, and a certain amount of urea (e.g., 4 mol/L) is added to prepare an antitumor drug preparation according to a conventional method.
  • urea e.g. 4 mol/L
  • the fusion protein concentrations of the above four drugs are generally in the range of 0.5-5.0 ⁇ 9 / ⁇ , and the dose is 4-12 g of fusion protein per g of nude mice.
  • Hela cells were cultured in RPMI 1640 medium, 10% calf serum, 37 ° C, 5% CO 2 culture. Receive The cultured cells were collected and injected into the roots of the hind limbs of nude mice at 2 X I0 6 / 0.1 mL / each.
  • nude mice After subcutaneous xenografts in nude mice were grown for 3 days (Hela), nude mice were randomly divided into experimental group and blank control group, with each group being thousands.
  • the blank control group was applied with PBS solution (or PBS glycerin solution) on the surface of the tumor
  • the experimental group was applied with the fusion protein PBS solution (or PBS glycerin solution) on the surface of the tumor; the application dose was controlled at 100 g/mouse as required.
  • the treatment was performed for 7 days, measured twice, and statistically analyzed for efficacy evaluation.
  • FIG. 12 The tumor growth curve of human cervical cancer Hela cell-bearing nude mice is shown in Figure 12.
  • Figure 13 shows the anti-tumor effect of PTD4-VP3 fusion protein on human cervical cancer Hela cell-bearing nude mice.
  • PTD4-VP3 fusion protein has the effect of inhibiting the tumor growth of human cervical cancer-bearing nude mice, and the fusion protein has the effect of treating cervical cancer.
  • mice Balb/C nude mice, male or female, 4-5 weeks old, weighing 15-20g. Purchased from the animal center of Hubei Provincial Epidemic Prevention Station;
  • SGC7901 cells were cultured in RPMI 1640 medium, 10% calf serum, 37 ° C, 5% C ⁇ 2 culture. The cultured cells were collected and injected into the roots of the hind limbs of nude mice at 2 X I0 6 / O.lmL.
  • PTD4-VP3 fusion protein was used to treat human gastric cancer.
  • SGC7901 cells were tumor-bearing in nude mice. The results are shown in the table below -
  • FIG. 14 The tumor growth curve of human gastric cancer SGC7901 cells in nude mice is shown in Figure 14.
  • Figure 15 shows the antitumor effect of PTD4-VP3 fusion protein on human gastric cancer SGC7901 cells in nude mice.
  • PTD4-VP3 fusion protein has the effect of inhibiting tumor growth in human gastric cancer-bearing nude mice, and the fusion protein has the effect of treating gastric cancer.
  • Example 12
  • HepG 2 cells were cultured in RPMM 640 medium, 10% calf serum, 37 ° C, 5% CO 2 culture. The cultured cells were collected and injected into the roots of the hind limbs of nude mice at 2 X I0 6 / 0.1 mL / each.
  • the tumor tumor block size of nude mice treated with PTD4-VP3 fusion protein in human hepatoma HepG 2 cells 7 days after inoculation is shown in the following table: Anti-tumor effect of white on human hepatocellular carcinoma HepG 2 cell-bearing nude mice.
  • the PTD4-VP3 fusion protein has the effect of inhibiting the tumor growth of human hepatocarcinoma-bearing nude mice, and the fusion protein has the effect of treating liver cancer.
  • the fusion protein formed by other protein transduction domains and CAV VP3 has the same therapeutic effect as the PTD4-VP3 fusion protein in treating tumors by dermal route.
  • the sequences of the nucleotides and amino acids involved in the patent application of the present invention are shown in the sequence listing, and the sequences in the table are:
  • Sequence 1 is a PTD4 coding strand DNA sequence
  • Sequence 2 is the amino acid sequence of PTD4;
  • Sequence 3 is the DNA sequence of chicken anemia virus vp3;
  • Sequence 4 is the amino acid sequence of chicken anemia virus V p3;
  • Sequence 5 is the DNA sequence of the PTD4-VP3 fusion protein
  • Sequence 6 is the amino acid sequence of the PTD4-VP3 fusion protein
  • Sequence 7 is the amino acid sequence of the PTD4-VP3 fusion protein digested by thrombin.
  • the references referred to in the patent application of the present invention are as follows:

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Abstract

La présente invention concerne une protéine de fusion utile pour le traitement de tumeurs, la protéine comportant un domaine de transduction des protéines (PTD) et la protéine VP3 du virus de l'anémie infectieuse du poulet. La protéine de fusion a la capacité de traverser efficacement les membranes biologiques, d'induire l'apoptose de cellules tumorales tout en laissant les cellules normales indemnes et d'éviter le risque de transduction de gènes hétérogènes dans les cellules. L'invention concerne également le procédé de préparation et les applications de la protéine de fusion dans la fabrication de médicaments anti-tumoraux, particulièrement lorsqu'elle est utilisée comme préparation percutanée pour le traitement de tumeurs.
PCT/CN2006/003636 2006-05-31 2006-12-28 Protéine de fusion ptd-vp3 utilisée comme médicament anti-tumoral et procédé de préparation WO2007140667A1 (fr)

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CNA2006100192401A CN101081871A (zh) 2006-05-31 2006-05-31 蛋白转导域4－凋亡素融合蛋白(PTD4－Apoptin)及其制法和应用

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CN101525388B (zh) * 2009-02-20 2012-02-01 中国人民解放军第四军医大学 特异性双链rna结合蛋白嵌合体及其在病毒感染性疾病中的应用
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