Preparation of 3D printed metoprolol tartrate chewable tablets and optimization of printing process / 药学学报

With the growing demand of personalized medicine for children, it is especially important to develop medicines for children. In this study, using metoprolol tartrate as model drug, we developed 3D printed chewable tablets suitable for children with automated dosage distribution using semi-solid extruded (SSE) 3D printing technology. Based on the quality by design concept, this study prepared a semi-solid material with good printability using gelatin as the substrate, constructed 3D models and printed tablets with the aid of computer-aided design. The printing parameters were optimized and determined as follows: print temperature of 35-37 ℃, print speed of 25 mm·s-1, fill rate of 15%, and number of outer profile layers of 2. Subsequently, the printing process and the quality uniformity of the tablets were verified, and a linear relationship between the dose and the number of model layers was obtained. Finally, 3D printed chewable tablets were superior in terms of appearance, dose accuracy and compliance compared with traditional split-dose commercially available tablets. In this study, 3D printed metoprolol tartrate chewable tablets with good performance were successfully prepared to address the personalized medication needs of pediatric patients.

Accelerated stability test of sustained-release thienorphine loaded microspheres for injection / 药学学报

In order to meet the clinical needs of long-acting sustained-release thienorphine, injectable thienorphine loaded microspheres were developed, and the accelerated stability study was carried out to explore the suitable storage and transportation conditions of the microspheres. Using poly(lactic-co-glycolic acid) (PLGA) as carrier material, 3 batches of microspheres were prepared in pilot scale with emulsion solvent evaporation method. By investigating the in vitro release of thienorphine loaded microspheres at 37, 45, 52, and 60 ℃, and applying the Arrhenius equation, the linear relationship between the release rate constant (lgk) and the temperature (1/T) was established to obtain the equation: lgk = -8.073/T + 24.35 (R2 = 0.985 3), which showed that the release of microspheres at high temperature can be used to predict the release in vitro at 37 ℃, and 52.0 ± 0.5 ℃ was selected as the accelerated release condition in vitro. The quality research methods were established to investigate the changes of critical quality attributes such as microsphere morphology, drug loading, particle size and distribution, polymer molecular weight, and the related substances under accelerated conditions. The difference factor f1 and similarity factor f2 were used to assess the similarity of release behavior under accelerated conditions. The results showed that under the accelerated experimental conditions of 25 ± 2 ℃ and relative humidity (RH) 60% ± 5%, the critical quality attributes of injectable thienorphine loaded microspheres had no significant change in 6 months, suggesting that the long-term storage condition could be 5 ± 3 ℃.

Preparation and evaluation of novel purine derivative F7 thermosensitive liposome / 国际药学研究杂志

Objective To prepare F7 thermosensitive liposome and evaluate its physicochemical properties, then investigate its cytotoxicity against tumor cells in vitro. Methods The F7 thermosensitive liposome was prepared by the pH gradient active drug loading method using dipalmitoyl phosphatidylcholine myristoyl lyso-phosphocholine and 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy (polyethylene glycol)-2000 as membrane materials. The encapsulation efficiency and drug loading were determined for the F7 thermosensitive liposome by HPLC. The phase transition temperature of F7 thermosensitive liposome was investigated by differential scanning calorimetry;the liposome morphology was observed by atomic force microscopy;the drug release of liposome was examined by dialysis;and the particle size and zeta potential were measured through Malvern particle size analyzer. The cytotoxicity of F7 and F7 thermosensitive liposome was determined by the MTT method, and the freeze-drying process was optimized using the designexpert software. Results The encapsulation efficiency of F7 thermosensitive liposomes was (97.56±0.22) ％, and the drug loading ratio was (1.51±0.01) ％. The phase transition temperature of F7 thermosensitive liposome was 39.9℃, the zeta potential was (-15.10±0.85) mV, the particle size was (86.94±1.21) nm, and the poly disperse coefficient was 0.17±0.01. Compared with the F7 injection, the F7 thermosensitive liposomes showed a stronger, dose-dependent inhibitory effect on the growth of lung cancer H1299 and breast cancer MCF-7 cells. The freeze-dried powder of liposomes dissolved well with the encapsulation efficiency of 95％ and the particle size of approximately 130 nm. Conclusion The F7 thermosensitive liposome prepared by the pH gradient active drug loading method has high encapsulation efficiency and good stability. The preparation method is simple and feasible for further development of the F7 preparation.

Preliminary processing, processing and usage of Dendrobii Caulis in history / 中国中药杂志

On account of the dense cuticles of the fresh stem and the light, hard and pliable texture of the dried stem, Dendrobii Caulis is difficult to dry or pulverize. So, it is very important to the ancient doctors that Dendrobii Caulis should be properly treated and applied to keep or evoke its medicinal effects. The current textual research results about the preliminary processing, processing and usage methods of Dendrobii Caulis showed that: (1) In history the clinical use of fresh or processed Dendrobii Caulis as teas and tinctures were very common. (2) Its roots and rhizomes would be removed before using. (3) Some ancillary approaches were applied to shorten drying times, such as rinsing with boiling mulberry-ash soup, washing or soaking with liquor, mixing with rice pulp and then basking, etc. (4) According to the ancients knowledge, the sufficient pulverization, by means of slicing, rasping, hitting or pestling techniques, was necessary for Dendrobii Caulis to take its effects. (5) The heat processing methods for Dendrobii Caulis included stir-baking, stir-frying, steaming, decocting and stewing techniques, usually with liquor as an auxiliary material. Among above mentioned, steaming by pretreating with liquor was most commonly used, and this scheme was colorfully drawn in Bu Yi Lei Gong Pao Zhi Bian Lan (Ming Dynasty, 1591 CE) ; moreover, decocting in advance or long-time simmering so as to prepare paste products were recommended in the Qing Dynasty. (6) Some different processing programs involving stir-baking with grit, air-tightly baking with ondol (Kangs), fumigating with sulfur, which appeared in modern times and brought attractive outward appearance of the drug, went against ancients original intentions of ensuring drug efficacy.

Current status and prospect of translational medicine in nanotechnology / 药学学报

Nowadays, nanotechnologies have shown wide application foreground in the biomedical field of medicine laboratory tests, drug delivery, gene therapy and bioremediation. However, in recent years, nanomaterials have been labeled poisonous, because of the disputes and misunderstandings of mainstream views on their safety. Besides, for the barriers of technical issues in preparation like: (1) low efficacy (poor PK & PD and low drug loading), (2) high cost (irreproducibility and difficulty in scale up), little of that research has been successfully translated into commercial products. Currently, along with the new theory of "physical damage is the origin of nanotoxicity", biodegradability and biocompatibility of nanomaterials are listed as the basic principle of safe application of nanomaterials. Combining scientific design based on molecular level with precision control of process engineering will provide a new strategy to overcome the core technical challenges. New turning point of translational medicine in nanotechnology may emerge.

Preperation and quality evaluation of vincristine sulfate thermosensitive liposomes / 中国药学杂志

OBJECTIVE: To prepare vincristine sulfate (VCR) thermosensitive liposomes, and evaluate its particle size and release characteristics in vitro and establish the methods for determination of content, entrapment efficiency and related substances.

Evaluation methods in vitro and in vivo for dry powder inhalers: Research advances / 国际药学研究杂志

Drug delivery to lungs is becoming an important means of administration of some drugs for lung disease and some protein drugs with systemic effects. This article introduces the inhalation devices and particle characteristics,the two main features of dry powder inhalers (DPI), then reviews the advances on evaluation methods in vitro and in vivo for DPI, such as next generation pharmaceutical impactor (NGI), the imitation between particles and cells using electrodynamic levitation, hydraulic lung and dry powder endotracheal insufflator device, providing references for research and development of DPI evaluation methods.

HLA-DR expression on regulatory T cells is closely associated with the global immune activation in HIV-1 infected subjects naïve to antiretroviral therapy / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The frequencies of regulatory T cells (Tregs) increased over the HIV infection but its counts actually decreased. We proposed that the decrease of Treg counts may cause the reduction of inhibitory effect and thereby account for the over-activation of Tregs during HIV infection. However, it remains unknown whether Tregs are also over-activated and thereafter the activation induced death may lead to the decrease of Tregs.</p><p><b>METHODS</b>Tregs were defined as CD4(+)CD25(+)CD127(lo/-) T cells. Eighty-one HIV-1 infected patients were enrolled in our study, and twenty-two HIV-1 seronegative donors were recruited as the control. The levels of HLA-DR on Tregs were determined by FACSAria flow cytometer.</p><p><b>RESULTS</b>Compared to HIV-1 seronegative donors, the levels of HLA-DR on CD4(+)CD25(+)CD127(lo/-) Tregs were significantly increased in HIV-1 infected patients, and its increase was positively associated with viral loads (r = 0.3163, P = 0.004) and negatively with CD4 T-cell counts (r = -0.4153, P < 0.0001). In addition, significant associations between HLA-DR expression on CD4(+)CD25(+)CD127(lo/-) Tregs and the percentages of HLA-DR, CD38, Ki67 expressing CD4(+) and CD8(+) T cells were also identified.</p><p><b>CONCLUSION</b>HLA-DR on Tregs is a good marker for viral replication and disease progression. The over-activation of Tregs might result in the decrease of Tregs.</p>

Expression and biological activity identification of recombinant Hap protein of NTHi / 南方医科大学学报

<p><b>OBJECTIVE</b>To express and purify Hap protein of nontypeable Haemophilus influenzae (NTHi) in prokaryotic system, and study its immunogenicity and adhesive activity.</p><p><b>METHOD</b>Hap protein was expressed in E.coli BL21 with pET32a (+)-Hap and purified by affinity chromatography. The adhesive activity of the recombinant Hap protein was observed in competitive adhesion assay using scanning electron microscope and bacterial counting. BALB/C mice were immunized intranasally with the purified recombinant Hap protein and cholera toxin B subunit (CT-B), and anti-Hap IgA and IgG were detected by enzyme-linked immunosorbent assay.</p><p><b>RESULTS</b>SDS-PAGE analysis showed a single band of the target protein, whose purity reached 85% according to the result of Gel analysis software. The concentration of the protein was 3.2 g/L after ultrafiltration and condensation. Competitive adhesion assay showed that compared with control group, the recombinant Hap protein significantly inhibited the adhesion of NTHi to ECM (P<0.01). Compared with Hap immunization alone, immunization with Hap combined with CT-B resulted in significantly higher titers of anti-Hap IgG and IgA in mice (P<0.05).</p><p><b>CONCLUSION</b>Highly purified recombinant Hap protein has been obtained in a prokaryotic system and shows good immunogenic and adhesive activities. These results will establish the basis for further study of NTHi vaccine.</p>

Construction of a eukaryotic expression vector of the gene encoding rat interferon-gamma-inducible protein and its expression in NIH 3T3 cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct an expression vector of the gene encoding rat interferon-gamma-inducible protein (IP-10) and identify its expression in NIH 3T3 cells.</p><p><b>METHODS</b>IP-10 gene was amplified by polymerase chain reaction (PCR) and inserted into the eukaryotic expression vector pcDNA3.1(+). After identification by PCR, restriction endonuclease digestion and sequence analysis, the recombinant expression vector pcDNA3.1(+)-IP-10 was transfected into NIH 3T3 cells via liposome. Immunofluorescence method was used to confirm the expression of pcDNA3.1(+)-IP-10 in the transfected NIH 3T3 cells. The expression of IP-10 protein in the supernatant of the transfected cells was examined by Western blotting.</p><p><b>RESULTS</b>PCR, restriction endonuclease digestion and sequence analyses confirmed successful construction of the recombinant vector pcDNA3.1(+)-IP-10. Immunofluorescence assay identified the expression of pcDNA3.1(+)-IP-10 in NIH 3T3 cells, and the expression of IP-10 protein was detected by Western blotting in the supernatant of the transfected cells.</p><p><b>CONCLUSION</b>A eukaryotic expression vector pcDNA3.1(+)-IP-10 has been successfully constructed, which provides the basis for investigating the therapeutic effect of IP-10 on Th1 type autoimmune disease.</p>

Effect of resveratrol on myocardial fibrosis in mice with chronic viral myocarditis / 中国当代儿科杂志

<p><b>OBJECTIVE</b>Some research has shown that resveratrol can ameliorate myocardial injury and improve cardiac function in mice with acute viral myocarditis (VMC), and can inhibit cardiac fibroblast proliferation and myofibroblast differentiation in vitro. This study was designed to investigate whether resveratrol has similar effects in the mouse model of chronic VMC.</p><p><b>METHODS</b>One hundred mice were inoculated with 0.3 mL of Coxsackievirus B3 1*106 TCID50. Thirty days later, the survivors (n=62) were used as a model of chronic VMC, and were randomly assigned to 4 groups: untreated VMC, and low- (10 mg/kg), middle- (100 mg/kg) and high-dose (1 000 mg/kg) resveratrol-treated VMC (once daily, for 30 days). Ten mice which received neither Coxsackievirus B3 nor resveratrol treatment served as the control group. After 30 days of resveratrol treatment, the mice were sacrificed. Serum concentrations of collagenous pre-peptides (PINP, PICP and PIIINP) were assessed using ELISA. Hematoxylin-eosin staining, picrosirius red staining and circularly polarized light were used to examine the histochemistry of myocardial collagen.</p><p><b>RESULTS</b>The myocardial collagen volume fraction in the high-dose (0.74+/-0.19) and the middle-dose (1.07+/-0.12) resveratrol-treated VMC groups was significantly lower than that in the untreated VMC (2.33+/-0.18) and the low-dose resveratrol-treated VMC (2.17+/-0.19) groups (P<0.05). Compared with the untreated VMC group, serum concentrations of PICP and PIIINP in the high-dose and the middle-dose resveratrol-treated VMC groups were significantly reduced (P<0.05), while PINP concentrations increased significantly (P<0.05).</p><p><b>CONCLUSIONS</b>Resveratrol can inhibit hyperplasia of myocardial collagen in the mouse model of chronic VMC, acting as an effective anti-fibrotic agent in the myocardium.</p>

Optimized isolation and purification of non-typeable Haemophilus influenzae Haps protein / 南方医科大学学报

<p><b>OBJECTIVE</b>To optimize the isolation and purification conditions for Hap(s) protein of non-typeable Haemophilus influenzae.</p><p><b>METHODS</b>Hap(s) protein was purified by ammonium sulfate precipitation, dialysis desalting and Hitrap weak cation exchange columns of CM Sepharose Fast Flow. The condition of the elution was optimized for pH and ionic strength, the absorbance at 280 nm of the elution samples were detected, and the targeted protein band in the collected samples was observed by SDS-PAGE electrophoresis.</p><p><b>RESULTS</b>The Hitrap ion exchange column was eluted with buffer 1, which resulted in a baseline distribution of absorbance at 280 nm. Buffer 2 elution of the column resulted in the presence of peak absorbance with trails, which was identified to be constituted by some low molecular weight bands by subsequent SDS-PAGE. In serial column elution with buffer 3 with different ionic strength, a peak absorbance was observed with the ionic strength of 100 mmol/L NaCl, and SDS-PAGE confirmed that the peak was generated by the target protein. No obvious peaks or bands in SDS-PAGE occurred with the other ionic strengths.</p><p><b>CONCLUSION</b>The pH of the buffer only affect the elution of the irrelevant proteins rather than the Hap(s) protein, and elution with the buffer containing 100 mmol/L NaCl can be optimal for eluting the Hap(s) protein.</p>

Cloning of ureI gene from Helicobacter pylori and its expression in E. coli / 南方医科大学学报

<p><b>OBJECTIVE</b>To clone the urea membrane channel gene (ureI) from Helicobacter pylori (Hp) for its expression in E. coli, and evaluate the expression conditions and immunological features of the fusion protein.</p><p><b>METHODS</b>ureI gene cloned by PCR from Hp was inserted into the plasmid pET32a (+) to construct the recombinant plasmid pET32a/ureI, followed by identification by BglII and HindIII digestion and sequencing. E. coli BL-21+(DE3) was transformed with pET32a/ureI to obtain the engineered bacterium BL21+/UreI, which was cultured at different temperatures and induced with 1.0 mmol/L IPTG for expression of the recombinant protein. The expressed proteins were identified by SDS-PAGE and analyzed by Pro-gel analyzer 4.0. Western blotting was performed to evaluate the immunogenicity of the expressed protein.</p><p><b>RESULTS</b>The cloned gene fragment was about 650 bp in length, and BglII and HindIII digestion of pET32a/ureI yielded a 650-bp band. Sequence analysis revealed that the cloned ureI gene contained 646 bp without reading frame alterations. Comparison against GenBank indicated a homology of 100% of the cloned gene with ureI gene of the corresponding Hp strains, and also one no less than 98.5% with ureI gene from other strains. The engineered E. coli BL21+/UreI could express recombinant UreI (rUreI) with His tag, and the target protein accounted for 20.2% of the total bacterial proteins after 1.0 mmol/L IPTG induction of the bacterium at 37 degrees C for 14 h. SDS-PAGE and Western blotting showed that the recombinant UreI protein was produced mainly in the inclusion bodies and fused with his-tag (rUreI/his), which could react with human anti-Hp and mAb to his tag but not with mAb to Hp UreB.</p><p><b>CONCLUSIONS</b>We have successfully cloned ureI gene and constructed the prokaryotic expression plasmid for efficient rUreI expression, and the fusion protein rUreI/his expressed in the inclusion bodies can react specifically with both Hp antibody and his-tag antibody.</p>

ST6Gal I siRNA and antisense oligonucleotide-mediated gene silencing lowers the invasiveness potential of colonic carcinoma cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the effects of small interfering RNA (siRNA) and antisense oligonucleotides (ASOs) targeting ST6Gal I on adhesion and invasiveness of human colonic carcinoma cell line SW480 over-expressing ST6Gal I.</p><p><b>METHODS</b>siRNA and ASOs targeting ST6Gal I were constructed and transfected into SW480 cells via lipofectmine 2000. SW480 cells were cultured and divided into 7 groups, namely the blank control group, liposome group, siRNA group (transfected with ST6Gal I siRNA), ASO(1) group (transfected with ST6Gal I ASO whose target site is different from the siRNA), ASO(2) group (transfected with ST6Gal I ASO targeting the same site as siRNA), siRNA+ASO(1) group (transfected with siRNA and ASO(1)), siRNA+ASO(2) group (transfected with siRNA and ASO(2)). RT-PCR was used to examine ST6Gal I mRNA expression following the treatment. Flow cytometry was used to examine the amount of alpha2,6-sialylation on SW480 cell surface. SW480 cell adhesion and invasiveness to the extracellular matrix (ECM) were analyzed using CytoMatrix kit and cell invasion assay kit, respectively.</p><p><b>RESULTS</b>The expression of ST6Gal I mRNA, the amount of alpha2,6-sialylation on the cell surface and cell adhesion and invasion to ECM decreased remarkably in groups siRNA, ASO(1), ASO(2), siRNA+ASO(1) and siRNA+ASO(2), all significantly lower than those of the blank control and liposome groups (all P<0.05), especially in siRNA+ASO(1) group. Significant difference was noted between siRNA+ASO(1) and siRNA groups (P<0.05), but not between siRNA+ASO(2) and siRNA groups, or between blank control and liposome groups (all P>0.05).</p><p><b>CONCLUSION</b>Chemically synthesized specific siRNA targeting ST6Gal I effectively inhibits SW480 cell ST6Gal I expression and leads to diminished cell adhesion and invasiveness to ECM, suggesting a combined effect of siRNA and ASO with different targeting sites.</p>

Effects of ST6Gal I antisense oligonucleotide-mediated gene silencing on cell adhesion and invasiveness of hela cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the effects of antisense oligonucleotide (ASODN) targeting ST6Gal I on cell adhesion and invasiveness of human cervical carcinoma cell line HeLa which over-expressed ST6Gal I .</p><p><b>METHODS</b>ASODN and sense oligonucleotide (SODN) targeting ST6Gal I were designed and constructed, and transfected into a cervical cancer cell line, HeLa, by lipofectmine 2000. HeLa cells were cultured and divided into 4 groups: blank control group, liposome group, SODN group and ASODN group. RT-PCR was used to examine the ST6Gal I mRNA expression. Flow cytometry was used to examine the amount of alpha2, 6-sialylation on the HeLa cell surface. The HeLa cell adhesion and invasiveness to extracellular matrix ( ECM) were analyzed by using CytoMatrixTM kit and cell invasion assay kit, respectively.</p><p><b>RESULTS</b>The expression of ST6Gal I mRNA in HeLa cells at 48 hrs after transfection in the ASODN group was significantly decreased in comparison with that in the blank control group, liposome group, and SODN group(P <0. 01). The amount of alpha2,6-sialylation on cell surface in ASODN group was significantly lower than that of the other 3 groups ( P <0. 05). The adhesion and invasiveness of the cells in the ASODN group decreased remarkably, both significantly lower than those of the other 3 groups ( all P < 0. 05).</p><p><b>CONCLUSION</b>Specific ASODN targeting ST6Gal I effectively inhibits HeLa cell ST6Gal I expression, decreases the amount of alpha2,6-sialylation on cell surface and leads to a decline of cell adhesion and invasiveness to ECM. This result also established a fine base for further studying on anti-tumor treatment with antisense oligonucleotide.</p>

Therapeutic effect of a new recombinant immunotoxin mMIP-1alpha-DT390 on experimental autoimmune encephalomyelitis / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the therapeutic effect of a new recombinant immunotoxin mMIP-1alpha-DT390 on experimental autoimmune encephalomyelitis (EAE).</p><p><b>METHODS</b>EAE was induced in the low-sensitive strain C57BL/6 mice with intraperitoneal injection of myelin basic protein (MBP) to simulate the human disease multiple sclerosis, followed by intramuscular injection of cationic liposome carrying the plasmid DNA SRalpha-mMIP-1alpha-DT390 in the leg muscle to elicit resistance to EAE development. The mice were then examined daily for clinical signs of EAE by an observer blind to the treatment protocol. For immunohistochemistry the mice were anesthetized and perfused with sterile PBS and paraformaldehyde, and the cerebrum, cerebellum, medulla and spinal cord were removed for preparation of serial sections. The mononuclear cells (MNCs) from the EAE mouse spleens were prepared for three-color flow cytometry analysis of the surface markers with appropriate antibodies following the BD Pharmingen cytokine staining protocol.</p><p><b>RESULTS</b>EAE model was successfully established by active MBP immunization in C57BL/6 mice. Administration of the immunotoxin mMIP-1alpha-DT390 significantly delayed the disease onset and lowered the mean clinical score for EAE as compared with the control mice. Immunohistochemistry demonstrated much less CCR5(+) infiltrating cells in the central nervous system in mMIP-1alpha-DT390-treated mice than in the control. The treatment also eliminated reactive T cells in the periphery blood without affecting the number of B cells.</p><p><b>CONCLUSION</b>The immunotoxin mMIP-1alpha-DT390 can attenuate the disease activity of EAE in mice, suggesting its potential use in the treatment of other autoimmune disorders.</p>

Protective effect of vitamin C on protein activity in plasma during virus inactivation / 中国实验血液学杂志

To determine whether addition of vitamin C (Vit C) to single-unit plasma could influence the efficacy of inactivating viruses and could maintain the activity of plasma proteins by methylene blue (MB)-light treatment. Vesicular stomatitis virus (VSV) Indiana strain was used as the indicating virus. Human plasma containing VSV was added with different concentrations of Vit C and final concentration 1 micromol/L MB and irradiated by fluorescence at an intensity of 40,000 lx, samples were collected at different times for detection. Cytopathic effect was used to test the effect of virus inactivation. A segment of the nucleic acid encoding capsid protein of VSV was amplified with RT-PCR. Some methods, such as the Clauss method, the one-stage method, microimmunoelectrophoresis, were used to investigate the changes of plasma components. The results showed that when the VSV plasma was added with 240 micromol/L Vit C and treated by MB-light irradiation for 60 min, the titer of VSV decreased by more than 8 lg TICD50/ml. Meanwhile, target segment amplification of VSV was also negative. The recovery rates of fibrinogen and coagulation factor VIII (FVIII: C) were 83.55% and 81.67% respectively, which had significant difference comparing with the routine MB-fluorescent light treatment. Most of plasma proteins were not affected significantly. No change in immunogenicity of these proteins was observed by using microimmunoelectrophoresis. It is concluded that virus inactivation is not influenced and plasma proteins are effectively protected by Vit C. Vit C can be used as a protector and is beneficial to improving the quality of plasma subjected to MB- photodynamic treatment.

Prokaryotic expression and identification of human papillomavirus type 16 E5 protein / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct the prokaryotic and eukaryotic expression vectors pET32/E5 and pcDNA3.1/E5 for transformation into E. coli BL21 and NIH(3)T(3) cells respectively to observe the expression of human papillomavirus type 16 E5 protein (HPV16 E5).</p><p><b>METHODS</b>HPV16 E5 gene was amplified by PCR from clinical isolates of HPV 16 and inserted into the plasmid pET32a(+) followed by digestion with BamH I and Hind III. The recombinant plasmid pET32/E5 was transformed into E. coli JM109 and selected with ampicillin. The positive clones containing the recombinant plasmid pET32/E5 were verified by restriction endonucleases BamH I and Xho and sequence analysis. The expression of HPV16 E5-TRX fusion protein in E. coli BL21(ED3) was identified by SDS-PAGE and Western blotting. The digestion product of BamH I and Xho was purified and inserted into the eukaryotic expression vector pcDNA3.1(+) to construct pcDNA3.1/E5, which was identified by sequencing and transfected into NIH3T3 cells. The NIH(3)T(3) cells with stable expression of HPV16 E5 were selected by G418 and confirmed by RT-PCR.</p><p><b>RESULTS</b>The pET32/E5 and pcDNA3.1/E5 vectors were constructed successfully. E.coli BL21(DE3) transformed by the recombinant plasmid pET32/E5 expressed HPV16 E5-TRX fusion protein efficiently. In the presence of 1 mmol/L IPTG at 28 degrees C, HPV16 E5-TRX recombinant protein accounted for about 10% of the total bacterial proteins. NIH3T3 cells stably expressing HPV16 E5 were harvested by selection with 250 g/ml of G418. HPV16 E5 gene from pcDNA3.1/E5-transfected NIH(3)T(3) cells was amplified by RT-PCR, and sequence analysis demonstrated the acquisition of the full-length gene fragment.</p><p><b>CONCLUSIONS</b>The prokaryotic and eukaryotic vectors for the HPV16 E5 gene have been successfully constructed. The acquisition of E .coli and NIH(3)T(3) cells with stable expression of HPV16 E5 protein may facilitate subsequent research of the biological properties and the transformation mechanism of HPV16 E5 protein on specific cells.</p>