重组人乳头瘤病毒58型L1蛋白在Sf9细胞中的表达条件优化

Objective To select the optimum conditions for amplification and expression of the recombinant human papillomavirus 58 (HPV58) LI gene using baculovirus-insect cell expression system. Methods The Sf9 insect cells at logarithmic phase was used to amplify virus at different multiplicities of infection (MOI). Titers of the virus were determined by plaque assay. The expression of recombinant protein was identified by indirect immunofluorescence assay, and the expression of recombinant protein at different MOI and time was determined by Western blot. Results Recombinant baculovirus containing HPV58 LI gene were amplified at MOI of 0.5 for 48 h when the Sf9 insect cells were at logarithmic phase, and the highest titers of the virus reached 5×108 pfu/ml. The optimum conditions for expressing the recombinant protein were that the recombinant baculovirus was inoculated at MOI of 5.0 and the protein was expressed within 72 h. Conclusion The optimum conditions for amplifying the virus and expressing the recombinant protein are determined.     

重组人乳头瘤病毒11型L1蛋白在昆虫细胞sf 9中的表达条件优化研究

Objective To determine the optimum conditions for expressing recombinant human papillomavirus 11 (HPVI1) L1 protein using haculovirus-insect cell expression system. MethodsThe expression of the recombinant protein was identified by indirect immunofluorescence assay, and the optimum conditions for expressing the recombinant protein at different multiplicities of infection (MOI) and time were determined by Western blot. Results The optimum conditions for expressing the recombinant protein were that the recombinant baeulovirus was inoculated at MOI of 2.0 when the density of sf 9 cell was 2.0 × 106/ml, and the protein was expressed through suspension culture within 96 h. Conclusions The optimum conditions for expressing the recombinant protein are determined, which is to provide reference for further expression and purification in large quantity.     

Human μ-opioid receptor overexpressed in Sf9 insect cells functionally coupled to endogenous G_(i/o) protein

Human μ-opioid receptor (HμOR) with a tag of six consecutive hisfidines at its carboxyl terminus was overexpressed in recombinant baculovirus infected Sf9 insect cells.The recombinant HμOR had high affinity to[~3H] diprenorphine and[~3H]ohmefentanyl (Ohm), and the binding was strongly inhibited by μ-selective agonists DAGO, Ohm, and morphine, but neither     

Silencing livin gene by siRNA leads to apoptosis induction, cell cycle arrest, and proliferation inhibition in malignant melanoma LiBr cells

Aim： The aim of the present study was to investigate the effects of silencing the livin gene by small interfering RNA （siRNA） on the expression of livin and the effects on apoptosis, cell cycle, and proliferation in human malignant melanoma LiBr cells. Methods： Three chemically-synthetic siRNA duplexes targeting livin were transiently transfected into the LiBr cells, and the effects on livin expression were detected both at the mRNA level by real-time RT-PCR and at the protein level by Western blotting. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling assay, flow cytometric analysis, and the expression of procaspase-3 and activated caspase-3 analysis by Western blotting. Cell cycle was analyzed by flow cytometry. Cell proliferation was determined by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide. Results： One of the 3 designed siRNA could effectively knock down the livin expression both at the mRNA and protein levels in dose- and time-dependent manners; 100 nmol/L with maximum downregulation on mRNA at 48 h, and on the protein at 72 h after transfection. Silencing livin could significantly induce apoptosis, arrest cell cycle at the Go/G1 phase, and inhibit proliferation in LiBr cells. Meanwhile, caspase-3 was activated. Conclusion： The livin gene could serve as a potential molecular target for gene therapy by siRNA for malignant melanoma.     

cDNA cloning, sequence analysis, and recombinant expression of akitonin beta, a C-type lectin-like protein from Agkistrodon acutus

AIM: To clone the cDNA of a new member of snake venom C-type lectin-like proteins, to study its structure-function relationships and to achieve its recombinant production. METHODS: PCR primers were designed based on the homology and cDNA was amplified by RT-PCR using total RNA from snake venom gland as the template.The PCR products were cloned into the plasmid pGEM-T and sequenced. The deduced protein sequence was analyzed with some bioinformatic programs. A recombinant expression plasmid was constructed using pBAD-TOPO as vector and transformed into E.coli TOP10 competent cells. RESULTS: A novel cDNA sequence encoding akitonin β was found and accepted by GenBank (accession number AF387100). Akitonin β consists of a typical carbohydrate recognition domain (CRD) of C-type lectins, and it is homologous with other snake venom C-type lectin-like proteins. It was predicted to be a platelet antagonist. Upon induction with arabinose rAkitonin β expressing in E coli was achieved at a high level (superior to 150 mg/L). The recombinant fusion protein exhibited inhibitory activities on rat platelet aggregation in vitro. CONCLUSION: A new member of snake venom C-type lectin-like proteins was discovered and characterized, and an efficient recombinant expression system was estab-lished for its production.     

Efficient delivery of human clotting factor IX after injection of lentiviral vectors in utero

AIM: To explore gene transfer feasibility for human clotting factor IX (hFIX) mediated by recombinant lentivirus in utero. METHODS: ICR mice fetus at 17-19 d gestation were received lentiviral vectors carrying hFIX cDNA under the control of liver specific promoter by intrahepatic injection. The expression and distribution of hFIX cDNA and possible immune responses against the hFIX were assessed by ELISA, PCR, RT-PCR, and immunohistochemistry, respectively. RESULTS: The serum hFIX protein were detected at different time points in all newborn mice, the highest level of hFIX was 50 μg/L and lasted for more than 30 d. Anti-hFIX antibody was not detected, hFIX cDNA was detected in liver, spleen, and heart. The expression of hFIX cDNA was only detected in liver. Besides, no germ line transmission was found at DNA and RNA levels, and no side effect associated with gene transfer was detected. CONCLUSION: The efficient delivery of hFIX can be achieved by prenatal gene transfer. It thus shows the feasibility of gene therapy for hemophilia in utero.     

Transfection of promyelocytic leukemia in retrovirus vector inhibits growth of human bladder cancer cells

Aim: To construct a recombinant retrovirus vector carrying human promyelocytic leukemia (PML) cDNA and identify its expression and biology role in bladder cancer UM-UC-2 cells for future gene therapy. Methods: PML full-length cDNA was inserted into the EcoR Ⅰ and BamH Ⅰ site of pLXSN vector containing the long terminal repeat (LTR) promoter. The vector was identified by restriction enzyme digestion and then transfected into PA317 packaging cell line by calcium phosphate coprecipitation. PML cDNA was detected by polymerase chain reaction (PCR) and the protein was identified by laser confocal microscopy and Western blot in bladder cancer cells, respectively. The morphology was observed by inverted phase contrast microscope, and MTF assay determined growth curve of the bladder cancer cells. Results: Restriction enzyme digestion proved that a 2.1kb PML cDNA was inserted into the pLXSN vector. PCR assay demonstrated that 304 bp fragments were found in UM-UC-2/pLPMLSN transfects. Laser confocal microscopy showed speck dots fluorescence in the UM-UC-2/pLPMLSN nucleus.A 90 kD specific brand was found by Western blot. MTT assay demonstrated the UM-UC-2/pLPMLSN bladder cancer growth inhibition. Conclusion: The retrovirus pLPMLSN vector was successfully constructed and could generate high effective expression of human PML in bladder cancer cell UM-UC-2, suggesting that PML recombinant retrovirus have potential utility in the gene therapy for bladder cancer.     

Overexpression of coxsackie and adenovirus receptor inhibit growth of human bladder cancer cell in vitro and in vivo

Aim： To study the effect of the overexpression of coxsackie and the adenovirus receptor （CAR） on the growth of the human bladder cancer cell in vitro and in vivo. Methods： A retroviral vector pLXSN-CAR expressing CAR was constructed and confirmed by restriction enzyme mapping. The pLXSN-CAR vector and con- trol vector pLXSN were transfected into the PT67 packaging cell line to generate retrovirus with high titer. The CAR-negative T24 cell was infected with the pLXSNCAR and the pLXSN retrovirus, respectively. The positive clone cells were selected with G418 for 2 weeks. The expression level of the CAR protein was detected by Western blot assay. T24 cell growth in vitro was determined by 3-（4, 5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay. Anchorage-independent growth was measured by soft-agar colony formation assay. In vivo cell growth was determined by a nude mice xenograft model. Results： The pLXSN-CAR vector containing full-length CAR cDNA was successfully constructed. Western blot analysis showed that a 46 kDa specific band was found in pLXSN-CA-transfected T24 cells. MTT assay identified the growth inhibition of T24/pLXSN-CAR cells. The cell colony forming ability of T24/pLXSNCAR cells was significantly lower than that of T24/pLXSN and parental T24 cells. There was a reduction in the tumor size in the T24/pLXSN-CAR group as compared with that of the T24/pLXSN group and parental T24 group. Conclusion： The overexpression of CAR in T24 bladder cancer cells can inhibit cell growth both in vitro and in vivo.     

Extraction and separation of total flavonoids from Flos Puerariae and its antioxidant activity

Objective To study the optimum extraction and separation process of total flavonoids in the flowers of Flos Puerariae and its antioxidative activity.Methods The total flavonoids were extracted with assistance of ultrasonic wave and the content was determined at 265 nm wavelength by Spectrophotometric method.Orthogonal experiment L9(34)was carried out to investigate the effects of concentration of solvent,the ratio of material to liquid,time length of extraction,and frequency of extraction on extraction results of total flavonoids from Flos Puerariae.The extracted solution was purified by petroleum ether and ethanol sequently.Under these conditions,the total flavonoids was eluted gradiently with mixed mobile phase of methanol-chloroform solution in the silica gel column system,and then determined by UV scanning and HPLC.Fenton reaction was used to produce and detect hydroxyl radicals(·OH),and pyrogallol system was used to produce and detect the superoxide radical anion(O2-·).Results The optimum conditions were as follows;using 40%(V/V)methanol as extractor with the ratio of material to liquid at 1∶30,and extracting for 2.5 h a time for 3 times.The extraction yield of total flavonoids was 13.6%.Six isoflavone compounds were isolated from the flowers of Flos Puerariae by the method of silica gel column chromatography.Antioxidative test results showed good performance of flavonoid scavenging capacity in both hydroxyl radical system and superoxide radical system and its IC50 was 7.65 μg·mL-1 and 0.18 mg·mL-1,respectively.Conclusions This study provided scientific basis for further development and utilization of Flos Puerariae and make preparation for later pharmacological research.