Targeting peptide-decorated biomimetic lipoproteins improve deep penetration and cancer cells accessibility in solid tumor

The limited penetration of nanoparticles and their poor accessibility to cancer cell fractions in tumor remain essential challenges for effective anticancer therapy. Herein, we designed a targeting peptide-decorated biomimetic lipoprotein (termed as BL-RD) to enable their deep penetration and efficient accessibility to cancer cell fractions in a tumor, thereby improving the combinational chemo-photodynamic therapy of triple negative breast cancer. BL-RD was composed of phospholipids, apolipoprotein A1 mimetic peptide (PK22), targeting peptide-conjugated cytotoxic mertansine (RM) and photodynamic agents of DiIC18(5) (DiD). The counterpart biomimetic lipoprotein system without RM (termed as BL-D) was fabricated as control. Both BL-D and BL-RD were nanometer-sized particles with a mean diameter of less than 30 nm and could be efficiently internalized by cancer cells. After intravenous injection, they can be specifically accumulated at tumor sites. When comparing to the counterpart BL-D, BL-RD displayed superior capability to permeate across the tumor mass, extravasate from tumor vasculature to distant regions and efficiently access the cancer cell fractions in a solid tumor, thus producing noticeable depression of the tumor growth. Taken together, BL-RD can be a promising delivery nanoplatform with prominent tumor-penetrating and cancer cells-accessing capability for effective tumor therapy.

Construction and identification of miR-186 overexpression lentiviral vector / 吉林大学学报(医学版)

Objective: To construct the miR-186 overexpression lentiviral vector and package the lentivirus, and to explore the infection efficiency and the expression level of miR-186 in the HEK293T cells. Methods: The Hsa-miR-186 precursor sequence was used as a template to design and synthesize the primer, and the the pre-miR-186 gene was amplified by PCR. The pre-miR-186 gene sequence was cloned into the lentiviral vector FV040 carrying EGFP/Puromycin cassette. The recombinant lentiviral vector was digested by EcoR I and Age I restriction endonuclease and confirmed by sequencing. The recombinant FV040 Vector and FV040 miR-186 were co-transfected into the HEK293T cells with the helper plasmids using Lipofectamine 2000, respectively; the FV040 Vector lentivirus (control group) and the FV040 miR-186 lentivirus (experiment group) were collected and used to infect the HEK293T cells 48 h after transfection, respectively. The green fluorescence distribution in the HEK 293T cells was observed 48 h after transfection, and the expression level of miR-186 was determined by real-time fluorescence quantitative PCR. Results: The sequencing analysis results indicated that the sequence of miR-186 overexpressing lentivirus was identical with the sequence of miR-186 published on GenBank. The titers in control group and experiment group were 6×108 TU · mL-1 and 5 × 108 TU · mL-1, respectively. The relative expression level of miR-186 in the HEK293T cells in experiment group (12. 640 0 ± 0. 788 4) was significantly increased by 15. 07 times (t=14. 72, P<0. 01) compared with control group (0. 838 7 ± 0. 145 6). Conclusion: The lentiviral vector which overexpresses miR-186 is constructed successfully and the miR-186 lentivirus is prepared. The HEK 293T cells are infected with miR-186 lentivirus successfully and the expression level of miR-186 in the HEK 293T cells is increased significantly.

Construction of human adenovirus type 4 vector expressing enhanced green fluorescence protein / 中华微生物学和免疫学杂志

Objective To prepare human adenovirus type 4 (Ad4) vector expressing enhanced green fluorescence protein (EGFP). Methods This study used a previously prepared plasmid pBRAd4 containing the whole genome DNA of Ad4-GZ01 strain. The Ad4 genome E3 region of pBRAd4 was deleted and replaced with the EGFP expression frame by conventional molecular cloning method. Then the recombi-nant plasmid was transfected into AD293 cells to rescue recombinant virus which was identified by sequen-cing,SDS-PAGE and ELISA. The purified virions were injected to mice and the induced immune responses were detected by ELISA and microneutralization test. Results The recombinant Ad4 vector rAd4EGFP ex-pressing EGFP was obtained and could be recognized and neutralized by monoclonal antibody MN4b and an-tisera against Ad4. The Ad4-specific and EGFP-specific antibodies with high titers could be detected in mice immunized with rAd4EGFP. Conclusion Human Ad4 vector expressing EGFP was successfully obtained and could be used in research on vaccine development,drug evaluation and transgene vector.

Construction of a low-pH-sensing system in Streptococcus mutans / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To construct a low-pH-sensing system in Streptococcus mutans (S. mutans) and to visually detect the pH in situ.</p><p><b>METHODS</b>Promoter of ureaseⅠ(PureⅠ) and green fluorescence protein (gfp) DNA fragments were amplified by polymerase chain reaction (PCR) from the genome of Streptococcus salivarius 57.I and S. mutans containing the gfp fragment. The two amplified DNA fragments were ligated together and further integrated into pDL278 to construct the recombinant plasmid pDL278-pureⅠ-gfp. This recombinant plasmid was then transformed into S. mutans UA159 cells. Subsequently, the intensity of the optical density per unit area of the low-pH-sensing system was measured and compared under different pH conditions and different processing times.</p><p><b>RESULTS</b>PureⅠ and gfp DNA fragments were amplified successfully with the correct molecule sizes (450 and 717 bp, respectively). The recombinant plasmid pDL278-pureⅠ-gfp was constructed and further verified by PCR and sequencing. The intensity of the optical density per unit area of the low-pH-sensing system increased with decreasing pH and increasing processing time.</p><p><b>CONCLUSIONS</b>A low-pH-sensing system was constructed successfully in S. mutans. Our research verified that pureⅠ of Streptococcus salivarius can function well in S. mutans as an acid induced promoter, and provided a new method of detecting the pH of plaque biofilms in situ.</p>

Engineering of Thiamin Pyrophosphate Fluorescent Biosensors Based on Ribozyme Switches in Mammalian Cells / 分析化学

Thiamin pyrophosphate (TPP) is a thiamine (vitamin B1) derivative and an essential cofactor in oxidative metabolism of the sugars,fatty acids and amino acids in living cells.By now,numerous TPP-dependent artificial riboswitch systems have been developed to regulate target gene expression but limited in bacteria,fungi or plant cells.Herein,the activating (switch-on) and inhibiting (switch-off) TPP-depended hammerhead ribozyme switches,which are from previous reported structures of prokaryotes screening,were investigated in mammalian cells.These ribozyme switches were inserted into the 3'UTR of the enhanced green fluorescence protein (EGFP) gene to construct the efficient ribozyme-based artificial switches through overlap extension PCR cloning.The HEK293 cells were transfected with the engineered ribozyme switches at increasing concentration of TPP.The EGFP gene-regulatory ability was analyzed with fluorescent microscope and flow cytometry.These TPP-inducible gene regulation devices showed the obvious ligand dose-dependency and excellent specificity.Two switch-on and one switch-off constructs demonstrated 3.1-fold or 1.9-fold increment and 2.3-fold reduction of EGFP level respectively with 150 μ mol/L TPP.The ligand-responsive ribozyme switches,by tuning the change of TPP concentration into the visual reporter genetic expression in cells,enable an efficient development of label-free,noninvasive and high-specific biosensors in living mammalian cells.

Construction and identification of eukaryotic expression plasmid carrying hTERT-P2A-EGFP / 吉林大学学报(医学版)

Objective:To construct the eukaryotic expression plasmid carrying hTERT-P2A-EGFP, and to explore its expression and transfection efficiency in the HEK293FT cells.Methods:The recombinant plasmid was constructed by using pBABE-puro-hTERT and pRRLSIN-cPPT-MSCV-EGFP plasmids.The hTERT,P2A,and EGFP genes were obtained using pBABE-puro-hTERT as template by PCR.And the correct hTERT was inserted into pRRLSIN-cPPT-MSCV-EGFP vector.Then the recombinant plasmid containing hTERT-P2A-EGFP gene was obtained and identified.The HEK293FT cells were transfected by the recombinant plasmid, and the expression of green fluorescence protein(GFP) was observed by fluorescence microscope.Results:The PCR results showed that the fragments of hTERT, P2A, and EGFP were 3 400, 110 and 720 bp.And the length of gene fragment(hTERT-P2A-EGFP)was 4 300 bp by enzyme digestion.The results of sequencing showed that the 1 547 site of the target gene was mutated.Using site-directed mutagenesis, the 1 547 site was successfully mutated.And the target gene sequence was completely identical with the sequence published in GenBank.The recombinant plasmid was transfected into the HEK293FT cells, and GFP was observed in the cells.The results of flow cytometry showed that the transfection efficiency of recombinant plasmid was 44.8%.Conclusion:The recombinant plasmid carrying hTERT-P2A-EGFP gene is successfully constructed, and it can be used for cell transfection.

Construction of hemolysin hlyA gene knocked out of cholera vaccine candidate and inserted green fluorescent protein gene / 实用医学杂志

Objective To knock down hemolysin hlyA gene and insert green fluorescence protein gene of vibrio cholerae vaccine candidate NFYY101. Methods Amplified fragments of hlyA gene upstream (hlyAup) and downstream (hlyAdown),lacz-GFPuv,and hlyAup-laczGFPuv-hlyAdown, and plasmids treated with specific enzymes were utilized to construct recombinant plasmids pUC18-hlyAup-laczGFPuv-hlyAdown and pCVD442-hlyAup-laczGFPuv-hlyAdown. Following the construction of the recombinant suicide plasmids ,a vaccine candidate was constructed by homologous recombination ,while SM10λpir carrying pCVD442- hlyAup-laczGFPuv-hlyAdown was utilized as the donor strain to transfect NFYY101. Results Construction of recombinant suicide plasmids pCVD442- hlyAup-laczGFPuv-hlyAdown was satisfactory that hemolysin hlyA gene was knocked out and green fluorescence protein gene was successfully inserted of vibrio cholerae vaccine candidate NFYY101. Conclusion Construction of the recombinant suicide plasmid pCVD442-hlyAup-laczGFPuv-hlyAdown successfully can be used for knocked out the hlyA gene and added green fluorescence protein gene as genetic marker into the chromosomal DNA of vibrio cholerae vaccine candidate.

Comparison of Lewis lung cancer cells and their derived cells / 中国实验动物学报

Objective By comparing the biological characteristics among Lewis lung cancer cells ( LLC) , LLC or-thotopic transplantation derivative cells ( R1-LLC) and R1-LLC orthotopic transplantation derivative cells ( R2-LLC) , we e-valuate their invasion and metastatic abilities in orthotopic transplantation models.Methods In vitro, we applied CCK8, clonogenic assay, and Transwell invasion assay to detect the proliferation ability, invasion ability and morphologic structures of LLC,R1-LLC, R2-LLC cells respectively, meanwhile observing morphologic structures by transmission electron microsco-py.In vivo, we constructed LLC, R1-LLC and R2-LLC orthotopic transplantation models.Herein, we observed their tumor growth and metastasis.The tumor formation rate and tumor-forming time were recorded for statistic analysis.Results In vitro:LLC, R1-LLC and R2-LLC cells showed no significant difference in proliferation ability ( P>0.05 ) , but significant differ-ences in invasion ability:R2-LLC>R1-LLC>LLC(P<0.05).In vivo:In the orthotopic group, the tumor formation rates of LLC, R1-LLC and R2-LLC cells were 66.67%、80%、93.33%(P <0.05).Conclusions In orthotopic implantation mouse models, R2-LLC cells present higher invasion and metastatic ability than R2-LLC and LLC cells.

Construction and Co-expression of Grass Carp Reovirus VP6 Protein and Enhanced Green Fluorescence Protein in the Insect Cells / 中国病毒学

Grass carp reovirus (GCRV), a disaster agent to aquatic animals, belongs to Genus Aquareovirus of family Reoviridea. Sequence analysis revealed GCRV genome segment 8 (s8) was 1296 bp nucleotides in length encoding an inner capsid protein VP6 of about 43kDa. To obtain in vitro non-fusion expression of a GCRV VP6 protein containing a molecular of fluorescence reporter, the recombinant baculovirus, which contained the GCRVs8 and eGFP (enhanced green fluorescence protein)genes, was constructed by using the Bac-to-Bac insect expression system. In this study, the whole GCRVs8 and eGFP genes, amplified by PCR, were constructed into a pFastBacDual vector under polyhedron (PH) and p10 promoters, respectively. The constructed dual recombinant plasmid (pFbDGCRVs8/eGFP) was transformed into DH10Bac cells to obtain recombinant Bacmid (AcGCRVs8/eGFP) by transposition. Finally, the recombinant bacluovirus (vAcGCRVs8/eGFP) was obtained from transfected Sf9 insect cells. The green fluorescence that was expressed by transfected Sf9 cells was initially observed 3 days post transfection, and gradually enhanced and extended around 5days culture in P1(Passage1) stock. The stable high level expression of recombinant protein was observed in P2 and subsequent passage budding virus (BV) stock. Additionally, PCR amplification from P1 and amplified P2 BV stock further confirmed the validity of the dual-recombinant baculovirus. Our results provide a foundation for expression and assembly of the GCRV structural protein in vitro.

Construction and expression of the eukaryotic expression plasmid of Human pEGFP/H2B / 重庆医科大学学报

Objective:To construct the eukaryotic expression plasmid of EGFP/H2B and express it in the Human embryo kidney cells(HEK293).Methods:Part H2B gene was obtained by RT-PCR from cultured HEK293 cell's whole RNA.The sequence encoding H2B was cloned into pEGFP-C1 to construct the eukaryotic expression vector.After confirmed by enzymatic digestion and sequencing,the recombination plasmid was transfected to HEK293 cells by lipofectamine technology for observation of the fusion protein's expression.Results:The recombination plasmid of pEGFP/H2B was constructed successfully.Compared with the dispersing green fluorescence among the whole cells in the control cells transfected with the mock plasmid(pEGFP-C1),the green fluorescence was concentrated in the nuclei of HEK293 cells transfected with pEGFP/H2B and distributed with the chromosomes in the mitotic cells.Conclusions:The successful construction of pEGFP/H2B recombination plasmids establishes an available survey system to study the chromosome segregation mechanism.

Construction and evaluation of a high effective GFP reporter plasmid for Streptococcus pneumoniae / 重庆医科大学学报

Objective: To construct a high effective GFP reporter plasmid for screening Streptoccus pneumoniae virulent genes by differential fluorescence induction. Methods: The SD-ENH-GFP region of plasmid pGreenTIR was cloned into a suicide plasmid pEVP3 which contains a cat gene encoding resistance protein to chloramphenicol,and a report plasmid pEVP3-SDGFP was constructed.To evaluate the function of this plasmid,a 500bp fragment of the pneumolysin gene (ply) of TIGR4 was coloned in the upstream of gfp and then was transformed into Streptococcus pneumoniae TIGR4. Results: The plasmid pEVP3-SDGFP could report the expression of ply both in vivo and in vitro,and was more effective than plasmid pEGFP-1 without SD and ENH sequence before gfp gene. Conclusion: Plasmid pEVP3-SDGFP can be used to construct the promoter-trap library which is needed in DFI.

Study on Expression,Purification of GFP-SA Recombine Protein and Anchoring Carcinoma Cells / 中国生物工程杂志

The GFP(green fluorescence protein)-streptavidin(SA) bi-functional fusion protein was generated and characterized in order to demonstrate novel platform for efficiently and durably modifying the cell surface with SA-tagged bi-functional proteins.The GFP-SA/pET24d construct was generated and expressed in BL21(DE3) host bacteria at the high level.The recombinant protein GFP-SA was purified through the Ni-NTA affinity chromatography,and then refolded.After biotinylation B16 tumor cells were modified with GFP-SA bi-functional fusion protein and then subjected to fluorescent microscopy and FACS analysis.The effect of surface modification on the viability and growth of B16.F10 tumor cells was evaluated by MTT staining.The GFP-SA recombinant fusion protein was expressed in BL21(DE3) at about 20 % of total bacterial proteins.The GFP-SA bi-functional fusion protein exhibited the bi-functionality,i.e.,SA-mediated high-affinity binding to biotinylated cell surfaces and GFP-emitted green fluorescence.The cell surface modification with GFP-SA bi-functional fusion protein did not affect the viability and growth of the modified B16.F10 tumor cells significantly.The GFP-SA bi-functional fusion protein was obtained and could be displayed efficiently on the surface of the biotinylated B16.F10 tumor cells through the specific and tight interaction between streptavidin and biotin,thus can be used as good trace protein and experimental control in the development of surface-modified tumor vaccine.

Cloning and Functional Analysis of a Promoter of ?-conglycinin Subunit Gene in Soybean / 中国生物工程杂志

A promoter fragment (7S?P ) of ? subunit gene was isolated by PCR from genomic DNA in various soybean accessions, including cultivars Nannong99-10,N2899,Nannong 88-1, and wild soybeans Jiangpu -1 and ZYD4174.The sequences of 7S?P fragment from these five soybean accessions shared 99% homology, this indicated that the promoter regions of ? subunit gene were conserved. Meanwhile , sequencing analysis showed that the 7S?P fragment contained several seed-specific motifs, such as RY motif, AGCCCA motif, ACGT motif and A/ T rich motif. The expression vector pBI121-7S?P was constructed with the 7S?P fragment (from Nannong99-10) and the GFP reporter gene for functional analysis. Arabidopsis plants were transformed by Agrobacterium mediated method. Southern blot results showed that the 7S?P had been integrated into the genome of Arabidopsis. Assay of GFP expression in the seeds of transgenic Arabidopsis was determined to identify the function of 7S?P promoter. The results showed that 7S?P was a seed-specific promoter.

Construction of Transferring Vector of Marek’s Disease Virus Expressing GFP Gene and Its Primary Application / 中国生物工程杂志

The expressing cassette, LoxP-CMV-gpt-IRES-LoxP( about 2.9kb), was amplified by PCR from a plasmid, pIRES-gpt, by use of the primers , which contained the loxP sites in 5' terminals, respectively. The loxP sites were designed into primers by the software of Primer primer 5.0. Then the cassette was cloned into the site of BalI in pBUS10 to obtain pUS-gptIRES(L). The sequencing analysis for pUS-gptIRES(L) indicated that two loxP sites with the same direction were correctly inserted into pUS-gptIRES(L).The gpt gene in pUS-gptIRES(L) was replaced by a fragment including the full length GFP gene as well as SV40 poly A sequence to get pUS-GFPIRES(L). pUS-GFPIRES(L) was transiently transfected into CHO cell lines, and then the green fluorescence could be seen, the results showed that GFP gene could be expressed correctly. Moreover, pUS-GFPIRES(L) was transfected into the CEF infected MDV CVI988 strain and recombinant virus was selected by the green fluorescence. The growth curve of virus showed the characteristic of recombinant virus was the same as that of CVI988 in vitro. These results give the basis for further studying the characteristic of MDV in vivo and the application of the Cre/LoxP system to MDV genome.

Effect of curcumin on sterol regulatory report system in HEK-293 / 中国药理学通报

Aim To investigate the lipid-lowering mechanism of Chinese Medicine curcumin.A sterol regulatory report system of LDLR gene expression in HEK-293 cell line was established.Method A green fluorescent protein(GFP) gene was constructed downstream of the sterol regulatory element-1(SRE-1) and the constructed plasmid was introduced into HEK-293 cell line.This SRE-GFP transfected 293 cells were treated with different concentration of curcumin for 2 days.The expression of GFP in the cells was measured by a fluorescence microscope and Flow Cytometry.Results The expression of GFP in SRE-GFP transfected 293 cells was obviously induced by curcumin.Conclusion Curcumin may positively affect the expression of LDLR gene by its influence on SRE-1.

Construction of the promoter indentifying plasmid with GFP reporter gene and identification of the promoter activity of HCV 5'- UTR / 重庆医科大学学报

Objective; To construct a promoter identifying plasmid with GFP as reporter gene, and then identify the promoter activity of HCV 5'- UTR with this construct. Methods: In order to construct the promoter identifying plasmid with GFP as reporter gene, the fragment of EGFP was obtained from the plasmid pEGFP - N1 by restrict enzymes digestion. And this fragment was combined with the larger fragment of plasmid PGL3 enhancer digested by the same enzymes. The fragment of HCV 5'- UTR was obtained by PCR amplification and inserted into the promoter identifying plasmid. The structure of constructed plasmid was confirmed by electrophoresis analysis and DNA sequencing. The function of construct was confirmed by lipofectamine - mediated transient expression in HepG2 cells. Results: DNA sequencing showed that the inserted fragment of the constructed plasmid was the same as the template HCV genome. The HepG2 cells transfected with promoter indentifying plasmid nearly didn' t express the reporter gene of GFP while the plasmid with HCV 5'- UTR could express the GFP gene. Conclusions; The results showed that the author successfully constructed the promoter identifying plasmid with GFP as reporter gene. The fragment of HCV 5'- UTR could behaviour as an eurakocyte promoter in HepG2 cells.

CONSTRUCTION OF AN INDUCIBLE VECTOR CARRYING GREEN FLUORESCENT PROTEIN / 解放军医学杂志

Objective To construct the inducible vector carrying green fluorescence protein(GFP). Methods We constructed an inducible vector pMD-GFP, including GFP cDNA, which allowed controlled expression of protein upon addition of 100 ?mol/L Zinc as an external inducer. The whole length of GFP cDNA were transfected into human hepatocellular cancer cells HCC-9204 by the method of lipofectin transfection. The expression of GFP was observed under fluorescence microscopy. Results The inducible vector carrying green fluorescence protein was successfully constructed. The observation under fluorescence microscopy showed that green fluorescence was spread in entire HCC-9204 cells transfected with GFP gene. Conclusion This new kind of the inducible vector could serve as a new tool and method for observing the growth and metastasis of neoplasm.

ClONING OF NP, P AND L GENE OF NEWCASTLE DISEASE VIRUS OF GOOSE ORIGIN AND IDENTIFICATION OF P GENE EXPRESSION / 微生物学通报

NP, P and L gene of Newcastle disease virus of goose origin were amplified and cloned into pGEM-T easy vector and then subcloned into pCI-neo expression vector respectively, the positive clones were identified by enzyme cutting, PCR and sequencing. GFP reporter gene was inserted into the downstream of recombinant expression plasmid of P gene, which of stop codon was deleted. The experiment of transfection of P and GFP recombinant plasmid on COS-1 cells and CEF showed that GFP gene expressed, and this demonstrated that P gene was also expressed. This research may be helpful for further study of reverse genetics and functional genome of NDV of goose origin.

Construction of the plasmid expressing gfp and MAD2 gene controlling separation of cell and its expression in HepG2 cell / 重庆医科大学学报

Objective: To establish an in vitro screening system for testing effects of MAD2 gene of embryonic cell genome.Methods: In order to construct the plasmid expressing MAD2-GFP fusion plasmid,the target gene fragment was obtained by RT-PCR amplification and inserted into pEGFP-N1 reporter vector.The structure of constructed plasmid was confirmed by electrophoresis analysis and DNA sequencing.The function of construct was confirmed by lipofectamine-mediated transient expression in HepG2 cells.Results: DNA sequencing showed that the inserted fragment of the constructed plasmid was the same as the template MAD2 genome.The HepG2 cells transfected with the constructed plasmid could express reporter gene of gfp.Conclusions: the plasmid expressing gfp gene controlled by MAD2 gene is successfully constructed and an in vitro testing system for evaluating founction of MAD2 gene in separation of cell is established.

Transient expression of BMP12 gene transfer to mesenchymal stem cells / 解放军医学杂志

Objective To investigate the feasibility of the committed differentiation of mesenchymal stem cells(MSCs) into tenocytes,the present study was carried out. Method NMP12 gene transfer to MSCs was performed with pEGFP C1 expressing vector by electroporation. Results Green fluorescence protein(GFP) expression was detected as early as 6h postelectroporation, peaked at 12h and could maintain 3 days. And then, GFP expression of some cells was weakened. Conclusion The results of the present study indicated that the successful transfection of MSCs was made, an the committed differentiation of MSCs into tenocytes was practicable