Biofunctionalization of commercially pure titanium with chitosan/hydroxyapatite biocomposite via silanization: evaluation of biological performances

Chitosan (CS) and hydroxyapatite (HA) bioactive molecules have been grafted onto commercially pure titanium surfaces (cpTi) to promote osteoblast adhesion and bone growth. The major challenge of this type of grafting is the attachment of the CS/HA biocomposite to the cpTi surface. In this study cpTi is biofunctionalized with CS/HA biocomposite material via silanization and the coated specimens were characterized using X-ray diffraction (XRD), scanning electron microscopy (SEM), water contact angle measurement and attenuated total reflectance-Fourier transform infrared (ATR-FTIR). The cpTi specimens were further evaluated for their in vitro bioactivity, hemocompatibility, protein adsorption and cell viability. The SEM micrographs showed uniform coatings adhered on cpTi specimens. The XRD and ATR-FTIR confirmed the presence of CS and HA on the cpTi specimens. The coated specimens showed improved in vitro bioactivity and hemocompatibilty along with enhanced adsorption of specific proteins. The cell viability studies showed non-cytotoxic nature of all the specimens and exhibited greater cell viability in the titanium CS/HA specimens. Hence, the studies showed that functionalized cpTi with covalent coating of CS/HA has significant potential in biomedical device implantation with improved bioactive properties.     

Potential of Biofermentative Unsulfated Chondroitin and Hyaluronic Acid in Dermal Repair

Chondroitin obtained through biotechnological processes (BC) shares similarities with both chondroitin sulfate (CS), due to the dimeric repetitive unit, and hyaluronic acid (HA), as it is unsulfated. In the framework of this experimental research, formulations containing BC with an average molecular size of about 35 KDa and high molecular weight HA (HHA) were characterized with respect to their rheological behavior, stability to enzymatic hydrolysis and they were evaluated in different skin damage models. The rheological characterization of the HHA/BC formulation revealed a G’ of 92 ± 3 Pa and a G″ of 116 ± 5 Pa and supported an easy injectability even at a concentration of 40 mg/mL. HA/BC preserved the HHA fraction better than HHA alone. BTH was active on BC alone only at high concentration. Assays on scratched keratinocytes (HaCaT) monolayers showed that all the glycosaminoglycan formulations accelerated cell migration, with HA/BC fastening healing 2-fold compared to the control. In addition, in 2D HaCaT cultures, as well as in a 3D skin tissue model HHA/BC efficiently modulated mRNA and protein levels of different types of collagens and elastin remarking a functional tissue physiology. Finally, immortalized human fibroblasts were challenged with TNF-α to obtain an in vitro model of inflammation. Upon HHA/BC addition, secreted IL-6 level was lower and efficient ECM biosynthesis was re-established. Finally, co-cultures of HaCaT and melanocytes were established, showing the ability of HHA/BC to modulate melanin release, suggesting a possible effect of this specific formulation on the reduction of stretch marks. Overall, besides demonstrating the safety of BC, the present study highlights the potential beneficial effect of HHA/BC formulation in different damage dermal models.     

Novel polysaccharides-based nanoparticle carriers prepared by polyelectrolyte complexation for protein drug delivery

Polyelectrolyte complexation, as one simple and promising method for preparing nanoparticles, is employed to find the use in the delivery of protein drugs. Using this method, we fabricated one kind of novel nanoparticles based on two natural polysaccharides, which are the negatively charged carboxymethyl pachyman (CMP) and the positively charged chitosan (CS). The major effect factors on the average particle size, polydispersity, and zeta potential of the nanoparticles were studied. The research indicated that the physicochemical properties of the nanoparticles were deeply affected by the molecular weight, concentration, and the ionic content of two polysaccharides. The mean particle size of CMP/CS nanoparticles was almost in the range of 100–200 nm for various preparation conditions. The morphology of nanoparticles characterized by a transmission electron microscope was spherical in shape with smooth surface structure. In order to study the feasibility of these nanoparticles as oral protein delivery carriers, the encapsulation efficiency of CMP/CS nanoparticles for bovine serum albumin (BSA) was evaluated for optimized condition. It turned out that the encapsulation efficiency of BSA-loaded CMP/CS nanoparticles varied from 30.1 to 52.9% depending on the initial loading concentration of BSA as well as the concentration of CMP and CS employed in particle formation, which indicated that the concentration of polymers and drugs were all contributed to the encapsulation efficiency of nanoparticles. This report opened up another interesting perspective to develop these natural polysaccharides with emerging new applications, which have great potentials in application in the nanoparticulate delivery system.     

Biocompatibility and antibacterial activity of chitosan and hyaluronic acid immobilized polyester fibers

Poly(ethylene terephthalate) (PET) fibers were treated with ⁶⁰Co‐γ‐ray and grafted with acrylic acid. The resulting fibers were further grafted with chitosan (CS) via esterification. Afterward, hyaluronic acid (HA) was immobilized onto CS‐grafting fibers. The antibacterial activity of CS against S. aureus, E. coli, and P. aeruginosa was preserved after HA‐immobilization. After immobilizing HA, the L929 fibroblasts cell proliferation was improved forCS‐grafting PET fiber. The results indicate that by grafting with CS and immobilizing with HA, PET fibers not only exhibit antibacterial activity, but also improve the cell proliferation for fibroblast. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 104: 220–225, 2007     

Interpolymer complex microparticles based on polymethacrylic acid‐chitosan for oral insulin delivery

In the present study, microparticles composed of polymethacrylic acid‐chitosan (PMAA‐CS) were prepared by a novel interionic gelation method. Free‐radical polymerization of methacrylic acid was carried out in the presence of CS, using a water‐soluble initiator, and application of these microparticles toward oral insulin delivery was evaluated. Microparticles obtained were characterized by scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) studies. From SEM studies, it was observed that microparticles had an aggregated morphology with size ～20 μm, while FTIR confirmed the presence of ionic interaction between PMAA and CS chains. Protein loading was done by diffusion filling method, and from in vitro release study, it was observed that insulin‐loaded microparticles displayed a pH depended release profile at alkaline/acidic pH. Microparticles exhibited sustained release of insulin for 3–4 h at neutral pH, and enzyme linked immunosorbent assay (ELISA) proved that encapsulated protein maintained 100% biological activity at neutral pH. Preliminary study suggests that these microparticles can serve as good candidate for oral protein delivery. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 99: 506–512, 2006     

Optimization of a Fabric Softener Formulation with an Electrochemical Sensor and Streaming Potential Measurements

An electrochemical, cationic, surfactant-selective sensor based on multiwalled carbon nanotubes (MWCNT) functionalized with a sulfate group and the cetylpyridinium ion (MWCNT–OSO₃⁻CP⁺) as a sensing material was used for optimization of the formulation of a fabric softener. Potentiometric titrations were performed and response measurements were obtained using four cationic surfactants (CS) of technical grade and four CS of analytical grade. The slope closest to Nernstian was obtained for di-(tallow carboxyethyl) hydroxyethyl methylammonium methosulfate (MAS) (59.5 ± 1.1 mV/decade of activity in water and 57.5 ± 1.3 mV/decade of activity in CaCl₂). When using CS as analytes in potentiometric titrations, the best accuracy (99.8%) was obtained when using MAS; therefore, it was chosen as the CS for the fabric softener formulation. Due to the better properties of fabric softeners with silicone in their formulations, four silicones at several concentration levels were used as potential additives. Based on the stability and viscosity of the system, the diquaternary polydimethylsiloxane (DPS) (w = 0.19%) was chosen for the fabric softener formulation. The pH did not significantly influence the potential when in the range of 3–8 or the recovery of potentiometric titrations when in the range of 3–7. The application profile of the CS was assessed through streaming potential measurements of reference fabrics in an electrokinetic analyzer. The obtained electrokinetic parameters indicated on lag in adsorption of model fabric softener (MFS) based on MAS (w = 9%), with the addition of silicone DPS (MFS 3), on cotton and polyester fabrics, but advantage in stability when compared with other MFS investigated.     

Carboxymethylcellulose–Chitosan‐coated microneedles with modulated hydration properties

Microneedles containing sodium carboxymethylcellulose (CMC) formulations were fabricated to include an external chitosan (CS) layer to modulate their hydration profile, an important parameter affecting their application as intradermal delivery devices and their storage. The microfabrication process was carried out under conditions that enabled the formation of polyelectrolyte complexes between these oppositely charged macromolecules. CMC–CS microneedles were characterized by water uptake in a humid environment, contact angle measurements, dissolution in aqueous solutions, and protein‐release profiles. The results demonstrate that the microneedles containing CMC–CS formulations displayed suppressed moisture sensitivity in water vapors compared to their unmodified CMC counterparts while the maintaining quick protein‐release characteristics required for their uses. This approach also showed the potential for sustained protein‐release applications, as the CMC–CS formulations could be combined in layers to fabricate multicompartment microneedle coatings with delayed release characteristics. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Effect of introduction of chondroitin sulfate into polymer-peptide conjugate responding to intracellular signals

We recently developed a novel tumor-targeted gene delivery system responding to hyperactivated intracellular signals. Polymeric carrier for gene delivery consists of hydrophilic neutral polymer as main chains and cationic peptide substrate for target enzyme as side chains, and was named polymer-peptide conjugate (PPC). Introduction of chondroitin sulfate (CS), which induces receptor-medicated endocytosis, into polymers mainly with a high cationic charge density such as polyethylenimine can increase tumor-targeted gene delivery. In the present study, we examined whether introduction of CS into PPC containing five cationic amino acids can increase gene expression in tumor cells. Size and zeta potential of plasmid DNA (pDNA)/PPC/CS complex were <200 nm and between -10 and -15 mV, respectively. In tumor cell experiments, pDNA/PPC/CS complex showed lower stability and gene regulation, compared with that of pDNA/PPC. Moreover, no difference in gene expression was identified between positive and negative polymer. These results were caused by fast disintegration of pDNA/PPC/CS complexes in the presence of serum. Thus, we suggest that introduction of negatively charged CS into polymers with a low charge density may lead to low stability and gene regulation of complexes.     

β‐cyclodextrin‐conjugated hyaluronan hydrogel as a potential drug sustained delivery carrier for wound healing

In order to develop a potential drug sustained delivery carrier suitable for wound healing, a series of β‐cyclodextrin conjugated hyaluronan hydrogels (β‐CD‐HA) with adjustable crosslink densities were synthesized and characterized, meanwhile the delivery kinetics and mechanism of diclofenac as a model anti‐inflammatory drug from these hydrogels were investigated. By controlling the feeding molar ratio of β‐CD/HA, a β‐CD substitution degree of 4.65% was obtained by ¹H‐NMR analysis. The incorporation of β‐CD modification had little effect on the internal porous structure, water swelling ratio, and rheological property of HA hydrogel, which however were influenced by the crosslink density. Although the crosslink density had an influence on the drug loading and release profile by altering the water swelling property, the interaction between β‐CD and drug was the primary factor for the high loading capacity and long‐term sustained delivery of diclofenac. The semiempirical equation fit showed that the release of diclofenac from HA‐based hydrogels followed a pseudo‐Fickian diffusion mechanism. By the aid of β‐CD and controlled crosslink density, a β‐CD‐HA hydrogel with a diclofenac sustained delivery period of over 28 days and desirable physicochemical properties was achieved, which will be a promising drug sustained delivery carrier for wound healing. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43072.     

A recombinant immunotoxin engineered for increased stability by adding a disulfide bond has decreased immunogenicity

Recombinant immunotoxins (RITs) are anti-cancer agents that combine the Fv of an antibody against cancer cells with a protein toxin from bacteria or plants. Since RITs contain a non-human protein, immunogenicity can be an obstacle in their development. In this study, we have explored the hypothesis that increasing stability can reduce the immunogenicity of a RIT using HA22-LR, which is composed of an anti-CD22 Fv fused to domain III of Pseudomonas exotoxin A. We introduced a disulfide bond into domain III by identifying and mutating two structurally adjacent residues to cysteines at sites suggested by computer modeling. This RIT, HA22-LR-DB, displays a remarkable increase in thermal stability and an enhanced resistance to trypsin degradation. In addition, HA22-LR-DB retains cytotoxic and anti-tumor activity, while exhibiting significantly lower immunogenicity in mice. This study demonstrates that it is possible to design mutations in a protein molecule that will increase the stability of the protein and thereby reduce its immunogenicity.     

Characterization of novel hyaluronic acid matrix systems for vaginal administration of metronidazole

Polymers such as Hyaluronic acid (HA), Polyethylene glycol‐400 (PEG‐400) and Xanthan Gum (XG) are promising in drug delivery applicationsbecause of their biomedical and pharmaceutical potential applications. In HA 2%‐PEG 400 systems, the effect of pH and PEG‐400 concentration were evaluated. The viscosity of HA‐PEG 400 formulations slightly increased with PEG‐400 concentration. Viscoelastic properties and shear thinning character was strongly dependent on pH. Structured systems were obtained at pH 3, with an increase of several orders of magnitude in zero‐shear viscosity values. When XG 1% structured system is added on HA (0, 0.5, and 2%) and PEG‐400 5%, a sharp increase of viscosity can be observed, obtaining a gel‐like behaviour for HA 0.5%‐XG 1%‐PEG 400 5% formulation. Finally, metronidazole release profiles in HA 2% formulations with different PEG‐400 concentrations at pH 4.5 were studied. At least 90% of metronidazole was releasedat 24 h. However, the addition of XG 1% to the HA (0.5 and 2%)‐PEG 400 5% systems delayed the drug release. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41313.     

Enhancement of bioactivity and bioavailability of curcumin with chitosan based materials

Curcumin (CUR) has been investigated for its poor accessibility to a site of action or absorption and rapid metabolism to cope with the limited medication and cure applications. This article reviews numerous approaches, such as encapsulated surfactant/polymeric micelles, liposomes, micro/nano-spheres, nano-suspensions/composites, nanocomplex, films, and hydrogels for effective transfer of CUR to target sites. Chitosan (CS), and chitosan derivatives have been found to enhance therapeutic efficacy of CUR. CS/modified-CS based alginate, cyclodextrin, starch, dextran sulfate, ZnO, phytosomes, and poly(butyl) cyanoacrylate drug delivery matrices improved bioavailability, prolonged drug loading and permeability, sustained release rate, improved solubility and stability (prevent metabolic degradation) of CUR, consequently promoting various clinical applications. CS based polysaccharide, protein, and metal oxide drug delivery nano formulations advantageously participated to improve biological activities of CUR. We have attempted to summarize these delivery approaches, and reviewed future trends/strategies to permit the introduction of CUR as practical therapeutic drug.     

A novel insulin oral delivery system assisted by cationic β‐cyclodextrin polymers

This work describes a new oral pharmaceutical formulation of insulin that is complexed with cationic β‐cyclodextrin polymers (CPβCDs), and then encapsulated into alginate/chitosan microspheres, which are prepared by ionotropic pregelation/polyelectrolyte method. CPβCDs were synthesized through a one‐step polymerization of β‐cyclodextrin (βCD), epichlorohydrin, and choline chloride. CPβCDs have enhanced ability to complex with insulin due to the assistance of their polymeric chains, as well as the electrostatic interactions between insulin (negatively charged while pH>5.3) and quaternary ammonium groups of CPβCDs. The noncovalent inclusion complex formed between CPβCDs and insulin was analyzed by Fourier transform infrared and fluorescence emission spectra. With the increase of zeta potential of CPβCDs from 1.8 to 14.2 mV, the insulin association efficiency (AE) of current system was increased from 55.2 to 71.8%, whereas the AE of insulin‐loaded microspheres at the same condition was only 50.7%. The cumulative insulin release in simulated intestinal fluid was also higher than that of the insulin‐loaded microspheres and βCD‐insulin encapsulated microspheres. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

pH-responsive nanoparticles shelled with chitosan for oral delivery of insulin: from mechanism to therapeutic applications

Despite advances in drug-delivery technologies, successful oral administration of protein drugs remains an elusive challenge. When protein drugs are administered orally, they can rapidly denature or degrade before they reach their targets. Such drugs also may not absorb adequately within the small intestine. As a protein drug for treating diabetes, insulin is conventionally administered via subcutaneous (SC) injection, yet often fails to achieve the glucose homeostasis observed in nondiabetic subjects. Some of this difference may relate to insulin transport: normally, endogenously secreted insulin moves to the liver via portal circulation. When administered subcutaneously, insulin moves through the body via peripheral circulation, which can produce a peripheral hyperinsulinemia. In addition, because SC treatment requires multiple daily injections of insulin, patients often do not fully comply with treatment. Oral administration of exogenous insulin would deliver the drug directly into the liver through portal circulation, mimicking the physiological fate of endogenously secreted insulin. This characteristic may offer the needed hepatic activation, while avoiding hyperinsulinemia and its associated long-term complications. This Account demonstrates the feasibility of using chitosan nanoparticles for oral insulin delivery. Nanoparticle (NP) delivery systems may provide an alternative means of orally administering protein drugs. In addition to protecting the drugs against a harmful gastric environment, the encapsulation of protein drugs in particulate carriers can avert enzymatic degradation, while controlling the drug release and enhancing their absorption in the small intestine. Our recent study described a pH-responsive NP system composed of chitosan (CS) and poly(γ-glutamic acid) for oral delivery of insulin. As a nontoxic, soft-tissue compatible, cationic polysaccharide, CS also adheres to the mucosal surface and transiently opens the tight junctions (TJs) between contiguous epithelial cells. Therefore, drugs made with CS NPs would have delivery advantages over traditional tablet or powder formulations. This Account focuses on the premise that these CS NPs can adhere to and infiltrate the mucus layer in the small intestine. Subsequently, the infiltrated CS NPs transiently open the TJs between epithelial cells. Because they are pH-sensitive, the nanoparticles become less stable and disintegrate, releasing the loaded insulin. The insulin then permeates through the opened paracellular pathway and moves into the systemic circulation.     

Preparation of chitosan bicomponent nanofibers filled with hydroxyapatite nanoparticles via electrospinning

Chitosan (CS) bicomponent nanofibers with an average diameter controlled from 100 to 50 nm were successfully prepared by electrospinning of CS and poly(vinyl alcohol) (PVA) blend solution. Finer fibers and more efficient fiber formations were observed with increased PVA contents. On this contribution, a uniform and ultrafine nanofibrous CS bicomponent mats filled with hydroxyapatite (HA) nanoparticles were successfully electrospun in a well devised condition. An increase in the contents of HA nanoparticles caused the conductivity of the blend solution to increase from 1.06 mS/cm (0 wt % HA) to 2.27 mS/cm (0.5 wt % HA), 2.35 mS/cm (1.0 wt % HA), respectively, and the average diameter of the composite fibers to decrease from 59 ± 10 nm(0 wt % HA) to 49 ± 10 nm (0.5 wt % HA), 46 ± 10 nm (1.0 wt % HA), respectively. SEM images showed that some particles had filled in the nanofibers whereas the others had dispersed on the surface of fibers, and EDXA results indicated that both the nanoparticles filled in the nanofibers and those adhered to the fibers were HA particles. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

Supramolecular Bioconjugates for Protein and Small Drug Delivery

Supramolecular conjugation techniques have been developed to produce novel nanosized systems by assembling materials with diverse physicochemical and biological features. These techniques have been adapted to obtain innovative bioconjugates to deliver drugs with poor biopharmaceutical properties and nano-devices with potential “theranostic” activity. Supramolecular drug delivery systems include polymer therapeutics such as drug–polymer bioconjugates, and colloidal carriers such as micelles, liposomes, polyplexes, and organic and inorganic nanoparticles. By virtue of their wide array of chemical composition and properties, polymers represent key elements for the construction of novel supramoelcular formulations. Polymer bioconjugation is a fledged technique for fabrication of protein–polymer conjugates. PEGylation, in particular, produces derivatives with enhanced pharmacokinetic, immunological, and stability properties as compared to the parent protein. Over the years, new methods have been set up to obtain site-directed polymer conjugation. In this review we report few grafting to and growing from PEGylation examples for the preparation of therapeutically effective protein bioconjugates. Supramolecular formulations with unique properties can be also obtained by assembling functional polymers, targeting agents, physicochemical modifiers, and biomodulators. These systems may be designed for disease tissue disposition and cell recognition/penetration. Cyclodextrins, for example, have been functionalized with polyethylene glycol and folic acid to produce tumor-targeted drug carriers. Interesting results have been obtained with this novel class of drug delivery systems. In addition, responsive polymers have been conjugated to gold nanoparticles to endow a new colloidal platform with triggerable cell disposition properties, which can be exploited either in biomedicine or diagnosis.     

Formation of coatings with tailored properties on polyperoxide-modified polymeric surfaces

Modification (peroxidation) of polymer surfaces, polypropylene, polyethylene, polyethylene terephthalate, nylon 6-6, poly(phenylene oxide), and ethylene–propylene copolymer were affected by the surface grafting of functional polyperoxides (FPPs) such as poly(5-tert-butylperoxy-5-methyl-1-hexene-3-yne-co-octylmethacrylate) (VEP-co-OMA), poly{N-[(tert-butylperoxy)-methyl]acrylamide-co-octyl methacrylate} (PO-co-OMA), and poly(tert-butylperoxy-methacrylate-co-octyl methacrylate) (PEst-co-OMA). The degree of surface modification was shown to be determined primarily by the structure of the polymer substrate. Using a peroxidized surface as an initiator of grafted copolymerization enabled the grafting of functional monomers (acrylic acid, acrylamide, 4-vinylpyridine, and hydroxyethyl methacrylate) and polysaccharides (heparin, dextran, etc.) and thereby imparted adhesive, antibacterial, haemocompatible properties to the polymer surface.     

Preparation and characterization of hyaluronan/chitosan scaffold cross‐ linked by 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide

A novel biocompatible scaffold was prepared by cross‐linking hyaluronan (HA) and chitosan (CS). The carboxyl groups of HA were activated by 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide (EDC) and then cross‐linked with amino groups of CS by forming amide bonds. The HA/CS scaffold thus prepared was characterized using Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM) and differential scanning calorimetry. FTIR spectra showed that the absorbance of the amide (1550 cm^?1) and carbonyl (1633 cm^?1) bond in the cross‐linked scaffold was stronger than that in HA or CS. SEM micrographs showed that the cross‐linked scaffold produced at low EDC concentration had an intertwisted ribbon‐like microstructure, while the product prepared at higher EDC concentration had a porous structure. The concentration of EDC in the reaction system greatly affected the structure and properties of the HA/CS scaffold. The prepared scaffold could strongly resist degradation by hyaluronidase, free radicals in vitro and stress. Copyright © 2007 Society of Chemical Industry     

Green synthesis of multifunctional carbon dots for anti-cancer and anti-fungal applications

Carbon dots (CDs) have become popular nanomaterials in biomedical and agricultural fields.Herein we synthesized multifunctional CDs which showed anti-cancer and anti-fungal activities.The low cytotoxi-city,stable fluorescence and high photothermal conversion efficiency enable the CDs with imaging-guided photothermal therapy.The CDs also exhibited intrinsic anti-fungal activity even at a low concen-tration,i.e.,40 mg·L-1 of CDs induced 20％ mortality in cucumber downy mildew.Moreover,the large π-conjugated nanostructure and the richness of amino and hydroxyl groups make them a powerful delivery platform for flumorph (a fungicide) with a high loading efficiency of 47.18％.Meanwhile,the heat con-verted from the light can accelerate the release of flumorph from CDs,and thus efficiently kill fungus.     

稳定表达甲型流感病毒血凝素蛋白的哺乳动物细胞系的建立

目的建立稳定表达甲型流感病毒血凝素(Hemagglutinin,HA)蛋白的哺乳动物细胞系。方法 PCR扩增流感病毒(A/PR/8/34)全长HA基因,并将其克隆入真核表达载体pcDNA5/FRT(pDF)中,构建重组表达质粒pDF-HA,将其与表达Flp重组酶的pOG44质粒共转染Flp-In-CHO细胞,通过体内同源重组使目的基因整合至宿主细胞染色体上。采用Hygromycin B持续压力筛选重组细胞系CHO-HA,通过间接免疫荧光法(IFA)和Western blot法检测HA蛋白的表达。重组细胞连续培养10代后,采用PCR和IFA法检测细胞中HA的基因遗传和蛋白表达的稳定性。结果重组表达质粒pDF-HA经双酶切及测序,证实构建正确;通过Hygromycin B抗性及IFA,共筛选出20株高表达HA蛋白的重组细胞株,Western blot结果进一步证实,HA蛋白在重组细胞中获得表达,并被切割成HA1和HA2蛋白;连续培养10代后,PCR与IFA方法分别检测到重组细胞HA基因和蛋白的表达。结论已成功建立了稳定表达甲型流感病毒HA蛋白的哺乳动物细胞系,为针对HA蛋白和流感病毒的免疫学检测以及HA蛋白的功能研究提供了靶细胞。