Effect of miR-204 targeted regulation of DVL3 gene in silica-induced mouse lung epithelial cells / 中华劳动卫生职业病杂志

Objective: To construct a recombinant lentiviral vector for mouse miR-204 overexpression, and to verify the targeted regulation of miR-204 and DVL3 in silica (SiO(2)) -induced mouse lung epithelial cells (MLE-12 cells) . Methods: In October 2019, the pre-miR-204 gene was amplified from the mouse genome by the polymerase chain reaction (PCR) method. After sequencing, the amplified product was cloned into the pLenti-CMV-EGFP lentiviral vector. The positive clones were identified by PCR screening and sequencing. The miR-204 overexpressed lentiviral vector was transfected into 293T cells, and lentiviral packaging and titer determination were performed. The experiment was divided into SiO(2) control group, virus control group, and miR-204 virus group, and the expressions of miR-204 and DVL3 gene were detected by real-time PCR. Results: The miR-204 lentiviral expression vector Lv-miR-204-5p was constructed and identified correctly by PCR and sequencing, and a virus dilution with a titer of 9.57×10(8) IU/ml was obtained. The results of real-time PCR showed that the expression of miR-204 in MLE-12 cells of the miR-204 virus group was higher than that of SiO(2) control group and virus control group, and the expression of DVL3 gene was lower than that of SiO(2) control group and virus control group, the differences were statistically significant (P<0.05) . Conclusion: Overexpression of miR-204 by lentiviral vector may inhibit the expression of DVL3 gene in silica-induced mouse lung epithelial cells.

Expression and function analysis of FaCO gene in Festuca arundinacea / 生物工程学报

Photoperiod plays an important role in transformation from vegetative growth to reproductive growth in plants. CONSTANS (CO), as a unique gene in the photoperiod pathway, responds to changes of day length to initiate flowering in the plant. In this study, the expression level of FaCONSTANS (FaCO) gene under long-day, short-day, continuous light and continuous darkness conditions was analyzed by real-time quantitative PCR. We constructed the over-expression vector p1300-FaCO and infected into Arabidopsis thaliana by Agrobacterium-mediated method. We constructed the silencing vector p1300-FaCO-RNAi and infected into Festuca arundinacea by Agrobacterium-mediated method. The expression of FaCO gene was regulated by photoperiod. The over-expression of FaCO promoted flowering in wild type of Arabidopsis thaliana under long day condition and rescued the late flowering phenotype in co-2 mutant of Arabidopsis thaliana. Silencing FaCO gene in Festuca arundinacea by RNAi showed late-flowering phenotype or always kept in the vegetative growth stage. Our understanding the function of FaCO in flowering regulation will help further understand biological function of this gene in Festuca arundinacea.

Effect of lentiviral vector mediated RNA binding protein quaking-5 on the proliferation and apoptosis of lung adenocarcinoma cell line H1299 / 解剖学报

To investigate the proliferation and apoptosis of lung adenocarcinoma cells line HI299 by the lentiviral vector mediated RNA binding protein quaking-5 ( QKI-5). Methods GV358 ( up-regulation ) and GV248 ( down-regulation) vectors were used to construct the lentiviral QKI-5 up-regulation vector and down-regulation vector, respectively. The vectors were transfected into 293T cells for lentiviral packaging and viral titer were then determined. Gene sequencing was performed to screen the sequence of vectors. Then Real-time PCR was used to evaluate the expression of QKI-5 mRNA and the proliferation of H1299 cells was examined by colony forming assay after transfection. The apoptosis of HI299 cells was determined by the detection of the expression membrane protein V (annexin V ) and propyl iodide ingot (PI) by flow cytometry. Pro-apoptotic protein Caspae-3 and Caspase-8 were evaluated by Western blotting. Results QKI-5 up-regulation and down-regulation lentiviral vectors were constructed successfully. Compared with the controls, the expression of QKI-5 mRNA of HI299 cells was up-regulated, the cell colony formation was decreased, and the early apoptosis of HI299 cells was increased with the over-expression of Caspase-8 after transfected with up-regulated vector, whereas transfecting with QKI-5 down-regulated vector had opposite effect. Conclusion Lenviral vector mediated QKI-5 could inhibit proliferation and promote apoptosis of lung adenocarcinoma cells through Caspase-8.

Prokaryotic expression, purification and functional identification of epidermal pattern factors in Arabidopsis thaliana / 生物工程学报

Stomatal density is important for crop yield. In this paper, we studied the epidermal pattern factors (EPFs) related to stomatal development. Prokaryotic expression vectors were constructed to obtain EPFs. Then the relationship between EPFs and hydrogen sulfide (H2S) was established. First, AtEPF1, AtEPF2 and AtEPFL9 were cloned and constructed to pET28a vectors. Then recombinant plasmids pET28a-AtEPF1, pET28a-AtEPF2 and pET28a-AtEPFL9 were digested and sequenced, showing successful construction. Finally, they were transformed into E. coli BL21(DE3) separately and induced to express by isopropyl β-D-galactoside (IPTG). The optimized expression conditions including IPTG concentration (0.5, 0.3 and 0.05 mmol/L), temperature (28 °C, 28 °C and 16 °C) and induction time (16 h, 16 h and 20 h) were obtained. The bands of purified proteins were about 18 kDa, 19 kDa and 14.5 kDa, respectively. In order to identify their function, the purified AtEPF2 and AtEPFL9 were presented to Arabidopsis thaliana seedlings. Interestingly, the H2S production rate decreased or increased compared with the control, showing significant differences. That is, EPFs affected the production of endogenous H2S in plants. These results provide a foundation for further study of the relationship between H2S and EPFs on stomatal development, but also a possible way to increase the yield or enhance the stress resistance.

Construction of prokaryotic expression and over-expression vectors of squalene epoxidase gene from Sanghuangporus baumii / 中草药

Objective To obtain prokaryotic expression and over-expression vectors of squalene epoxidase (SE) gene from Sanghuangporus baumii. Methods The entire protein-coding cDNA of SE was cloned into the expression vector pET-32a (+). Then the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) cells. SDS-PAGE was used to investigate the situation of expression after IPTG induction for 2-10 h. Additionally, primers were designed according to the gpd promoter sequence of Lentinula edodes in GenBank, and the gpd promoter fragment was obtained by PCR. Subsequently, the plant binary expression vector pCAMBIA1301 was selected as the basic vector, and then the 35 S promoter replaced with L. edodes gpd promoter through enzyme digestion and connection. Finally, the coding region of SE was cloned to the downstream of the gpd promoter to construct over-expression vectors. Results The prokaryotic expression vector pET-32a-SE was successfully obtained. SDS-PAGE results showed a significant protein band was found in the vicinity of the relative molecular weight of approximately 55 000, consistent with molecular weight of the predicted protein. Moreover, the over-expression vector pCAMBIA1301-gpd-gpd-SE was constructed successfully through different detection ways. Conclusion These results lay the foundation for the further study of SE in triterpenoid biosynthesis pathway of S. baumii.

Construction of Prokaryotic Expression Vector,Expression,Purification,and Identification of Fusion Protein,TAT-RabGEF1 / 中山大学学报(医学科学版)

[Objective]To construct recombinant expression vector PET28a-TAT-RabGEF1,express,purify fusion pr-otein effectively in E.coli Rosetta(DE3),and investigate its transmembrane effect in vitro on P815 cells.[Methods]With cDNA in rats′tissues as the template,two primers containing the TAT sequence and two designed enzyme restriction cutting sites were designed. TAT-RabGEF1 fragment was amplified by PCR,and its product was inserted into PET-28a vector to construct recombinant plasmid PET28a-TAT-RabGEF1 prokaryotic expression vector. The recombinant vector was trans-formed into E.coli Rosetta(DE3)and express fusion proteins.The protein products were purified by affinity chromatography. The efficiency of the transduction of fusion protein into P815 cells were detected by immunofluorescence and analyzed by fluo-rescence spectro-photometer,with using methods of CCK-8 to analyze the cells viability after transduction of different con-centrations of fusion protein.[Results]The recombinant vector PET28a-TAT-RabGEF1 was constructed and the fusion pro-tein TAT-RabGEF1 was successfully expressed in E.coli Rosetta(DE3). By western blotting and SDS-PAGE we can see that the protein products′ relative molecular mass was about 57 ku,which was consistent with the target one,TAT-Rab-GEF1.The immunofluorescence results suggested that the fusion protein had the ability to transduct into P815 cells,and sat-uration of fusion proteins to be transducted into cells was 1 μmol/L.[Conclusion]Constructed recombinant vector PET28a-TAT-RabGEF1 and expressed fusion protein,TAT-RabGEF1,which verified the TAT′s ability of transduction. And it would build a solid technical foundation for the following research on the effect of RabGEF1′s on the activation of mast cells.

Construction of lentiviral vector-mediated Cx32 stably over-expressed Huh7 cell line and its effect on cell proliferation / 中国药理学通报

Aim To construct a lentiviral vector for stable delivery of the connexin32 (Cx32) gene in human hepatocellular carcinoma cell line Huh7,and also to detect its effect on cell proliferation.Methods Human Cx32 gene sequence was obtained by whole gene synthesis and amplified by PCR,and was then inserted into LV5-GFP lentiviral vector to construct the recombinant plasmid LV5-GFP-hCx32.Following identified by restriction endonuclease digestion and DNA sequencing,this plasmid together with lentiviral package plasmid was transfected into 293T cells to produce the lentiviral particles,and the viral titer was assessed under fluorescent microscope.Targeted Huh7 cells were infected with the lentivirus (LV5-hCx32 and LV5-NC),and the infected cells after selection with puromycin were amplified and cultured.The expression and localization of Cx32 were detected by real-time RT-PCR,Western blot and immunofluorescence assay,respectively.The gap junction (GJ) function between adjacent cells was measured by dye transfer assay.The Huh7 cell proliferation capacity was determined by MTT and colony formation assays.Results The results of double enzyme digestion and DNA sequencing proved that the recombinant lentiviral vector LV5-GFP-hCx32 was successfully constructed.After packing in 293T cells,the recombinant lentivirus LV5-GFP-hCx32 with virus drops to 3 × 1011 TU · L-1 was obtained.Huh7 cells transiently infected with the lentivirus LV5-GFP-hCx32 remarkably over-expressed Cx32 at both mRNA and protein levels.Moreover,Cx32 expression was also significantly up-regulated in stably transfected Huh7 cells,and the presence of enhanced functional GJ in intact cells could be detected due to an increased amount of Cx32 protein along the plasma membrane at cell-cell contacts.Compared to LV5-NC group,the proliferation ability in Huh7 cells with recombinant Cx32 declined (P ＜ 0.05).Conclusions The lentiviral vector over-expressing Cx32 gene is successfully constructed,and can stably transfect Huh7 cells to yield sustained over-expression of exogenous Cx32 gene,thus eventually inhibits cell proliferation.

Construction and identification of shRNA lentiviral vectors targeting histone lysine transferase KAT6B gene / 实用肿瘤学杂志

Objective The aims of this study were to construct short hairpin RNA(shRNA)lentiviral vector in breast cancer T47D cells,to carry out RNA interference on lysine acetyltransferase 6B(KAT6B/MORF)gene,to down-regulate its expression and to explore its function.Methods Two pairs of single-stranded short hairpin RNA(shRNA5 and shRNA8)and the corresponding control sequences(Scramble5 and Scramble8)were synthesized based on the CDS of KAT6B gene.Polymerase chain reaction(PCR) was used to amplify double-stranded and ligated with the entry vector(pENTR/pSM2(CMV)GFP),which were subjected to a doub-le digestion(EcoRl and Xhol)linearization and homologous recombination with the entry vector(pENTR/pSM2(CMV)GFP)to obtain an entry clone containing the desired fragment.The target fragment was recombined onto the target vector(pLenti x1 puro DEST)via the LR cloning reaction of the Gateway system.The lentiviral packaging plasmids were co-transfected into HEK-293T cells with two pairs of target plasmids. The supernatant of HEK -293T cells was collected and transformed into T47D cells. The expression of KAT6B protein was detected in T47D cells by Western blot.Results The single colony obtained from the transformation was identi-fied by sequencing,which was consistent with the target sequence,indicating that the lentiviral vector had been successfully construc-ted.The expression of KAT6B protein was significantly lower in the shRNA KAT6B group than that in the control group,which indica-ted that the constructed gene silencing vector could play a role in the KAT6B gene in T47D cells.Conclusion The shRNA lentiviral gene silencing vectors of KAT6B were constructed and identified in T47D cells,which indicated that the foundation for further study

Construction of fused recombinant vector of EGFR gene containing G719S and T790M by overlap PCR / 临床检验杂志

Objective To construct the recombinant pMD19-exon18-exon20 plasmid containing locus G719S and T790M of EGFR gene associated with cervical cancer,which may provide a template for preparing the mutant recombinant vector of EGFR gene.Methods Using the healthy human genome DNA as templates,the segments of exon 18 and exon 20 of EGFR gene were amplified by two pairs of specific primers which were designed based on the sequences of overlapping and complementary area.The amplified segments were linked by overlap PCR.The products of linked exon18-exon20 were further inserted into the vector pMD19-T.The constructed recombi nant pMD19-exon18-exon20 plasmid was finally transformed into competent cells E.coli DH5α and then identified by PCR with bacterial solution and genome sequencing.Results The amplified fragments of exon18 and exon20 were clearly appeared at 778 bp and 596 bp and the fused product of exon18-exon20 was showed at 1 374 bp on agarose gel electrophoresis.The recombinant plasmid of fusion EGFR gene was consistent with the expected results via bacterial PCR assay and DNA sequencing.Conclusion We successfully fused the segments of exon18 with exon20 and constructed the recombinant expression vector of EGFR gene by using overlap PCR method.

Expressions of CD49f splicing isoforms in different cell lines / 基础医学与临床

Objective To compare the expression of CD 49f splicing isoforms in different human stem cells and colon cancer cell lines , and construct lentiviral vectors overexpressing specific isoform .Methods The expression of CD49f isoforms in different cells was detected by RT-PCR and real-time PCR.The lentiviral vectors overexpressing CD49f isoforms were constructed by molecular cloning method .The overexpression of CD49f in colon cancer cell line HT29 was confirmed by FCM, Western blot and real-time PCR, the invasive ability was examined by transwell assay.Results CD49f splicing isoform B was highly expressed in H 9 human embryonic stem cell line , while iso-form A expressed in epithelial cells .Both isoform A and B were expressed in mesenchymal cells .CD49f isoforms A and B were also expressed in colorectal cancer cell lines , while HT29 and HCT116 showed higher expression of iso-form A than isoform B , HCT8 and LoVo showed higher expression of isoform B than isoform A .Overexpression of CD49f isoform B greatly increased the invasive ability of HT 29 cells, while isoform A showed no effect .Conclusions The expressions of CD49f splicing isoforms A and B in different types of cells are significantly different , which sug-gests that CD49f isoforms play different biological functions in cells .

A new method of RNAi vector construction and its application of secondary metabolism regulation of plants / 中草药

RNA interference (RNAi) technology has strong specificity, high inhibitory efficiency, large-scale operation application, and good repeatability, and it can perform Mendelian mode of inheritance in plants, which is widely used in the validation of gene function and metabolic regulation. In plant metabolic engineering, the RNAi method can inhibit the synthesis of product, change the distribution of metabolic flux, and regulate the synthesis of the target product. This review summarized the current progress and study strategy of RNAi in plant metabolic regulation. RNAi mechanism and characteristics, optimal strategy of vector structure, efficient RNAi vector construction method, and application progress in the regulation of metabolism were reviewed, in order to lay the theoretical foundation and technical reference of RNAi technology of metabolic engineering control.

Expression of lentivirus-mediated rat prodynorphin gene in bone marrow mesenchymal stem cells / 医学研究生学报

Objective Dynorphins have advantages in powerful analgesic effect, high safety, no respiratory depression and no addiction, which is the emphasis of analgesic research at present. The aim of the article was to explore the expression of lentivirus-mediated rat prodynorphin gene in bone marrow mesenchymal stem cells( BM-MSCs) and contribute to the subsequent studies on bio-logical analgesia in cancer pain of rat model. Methods BM-MSCs were isolated and proliferated using the adherence screening meth-od, and further identified by flow cytometry, adipogenic and osteogenic differentiation experiments. The PDYN lentiviral vectors in rats were transfected into BM-MSCs after construction. The expression of green fluorescent protein (GFP) was detected under inversion fluo-rescence microscope and the best multiplicity of infection ( MOI ) of virus was screened by western blot. There are three groups in the ex-periment: blank group, experimental group ( PCDH-CMV-PDYN-EF1-copGFP ) and empty vector group ( PCDH-CMV-MCS-EF1-copGFP). PYDN gene was determined by qPCR and western blot, while DYN protein was detected by immunochemical method.Results BM-SMCs were in longspindle-shape and fibrocyte-like adherent growth, most in expression of CD29, CD44 and CD90, and a few in CD45. The oil red-O staining of the induced cells by adipogenic differentiation was positive. The mineralized nodules formed in the induced cells by osteogenic differentiation were orange after alizarin red staining. Flow cytometry detection showed the positive rates of CD29, CD90, CD44 and CD45 were respectively (99.80±0.19)%, (99.62±0.24)%, (96.86±1.27)%, (0.82±0.06)%, while after transfection the positive rates were (99.59±0.34)%, (98.06±1.27)%, (95.23±0.71)%, (10.23±0.59)%, representing no sig-nificant difference before and after PDYN transfection. Lentiviral vector of PCDH-CMV-PDYN-EF1-copGFP was successfully construc-ted after the identification of PCR amplification, cloning and sequencing. The titer of recombined lentiviruses was 5×106IU/mL. The best MOI of lentiviruses was 100 according to the results of GFP and western blot. Western blot and qPCR suggested PDYN gene signif-icantly increased in BM-MSCs after lentiviral transfection ( P<0.05) , and immunohistochemical staining indicated DYN protein also in-creased greatly. Conclusion BM-MSCs are successfully cultured and the overexpressed rat PDYN gene lentivirus vector is also suc-cessfully constructed;PDYN gene is highly and stably expressed and DYN protein is secreted in BM-MSCs.

Construction and identification of lentiviral vector over-expressing Staphyloco-ccus aureus enterotoxin C3 / 中国免疫学杂志

Objective:To construct the lentiviral vector over-expressing Staphylococcus aureus enterotoxin C3 and detect the expression of target gene in vitro.Methods:SEC3 gene were amplificatied by polymerase chain rcaction( PCR).The GV365 lentiviral vectors were digested by AgeⅠenzyme,which was linked to SEC3 gene and then constructed the GV365-SEC3 lentiviral vetor.Positive clones of vectors were identificd by PCR.Then the positive lentiviral vectors were transfected into 293T cells for lentivirus package.The expression of lentiviral vectors was tested by observating cell fluorescence and Western blot.The virus titer was determined by HIV-1 p24 ELISA.Results: SEC3 gene was amplified and successfully bound to the GV365 lentivirus vectors.The sequences of the recombinant plasmid were confirmed correct by PCR and DNA scqucncing.A large mass of green fluorescent cells were observed after transfecting.And the resulting size of 29 kD protein band of protein electrophoresis, which was consistent with the target gene protein.Viral vector titer was 5×108 TU/ml by ELISA detection.Conclusion: Lentiviral vector over-expressing Staphylococcus aureus enterotoxin C3 was successfully constructed,laid the foundation of observing its effect and mechanism against to tumor in vivo and in vitro for later research.

Construction of expression vector of PgMYB4 gene in Panax ginseng and analysis on drought resisting / 中草药

Objective: To construct the expression vector of PgMYB4 gene in Panax ginseng and study its function of the drought resisting in Arabidopsis thaliana. Methods: A P. ginseng gene PgMYB4 was cloned by RT-PCR analysis, further, recombinant plasmid vector with PgMYB4 was transformed into wild-type plants of A. thaliana by Agrobacterium tumefacies-mediated Floral Dip method. Transgenic A. thaliana with the expression of PgMYB4 was obtained, further compared with wild-type plants of A. thaliana, their determination of physiologic index related to drought stress was detected. Results: The cDNA (named a PgMYB4) contained a 735 bp open reading frame, encoded 245 amino acids and the predicted molecular weight was 27.914 KDa; Under the condition of drought stress, the growth of transgenic A. thaliana was obviously better than wild-type A. thaliana. Compared with wild-type A. thaliana, the decreased range of relative chlorophyll content in the leaves of transgenic plants of A. thaliana was smaller and the proline content of transgenic plants of A. thaliana increased significantly. The water loss of transgenic plants of A. thaliana was less than that of Wild-type transgenic plants of A. thaliana. Conclusion: Ginseng PgMYB4 gene has the ability of resistance to drought stress.

Prokaryotic expression and purification of Aquilaria sinensis (Lour.) Gilg AsMYC2 protein / 药学学报

The MYC2 transcription factor is a member of the important plant bHLH transcription factor families, and it is also the core regulatory elements in jasmonate (JA) signaling pathway. However, there is a little information about AsMYC2 gene in Aquilaria sinensis. In this study, with the total RNA isolated from A. sinensis leave as template, the full-length coding sequence (CDS) of AsMYC2 gene was amplified using RT-PCR method and subcloned into pGEX-4T-1 vector by the gene recombination technique. The recombinant vector pGEX-4T-1-AsMYC2 was verified by restriction enzyme digestion and nucleotide sequencing, and was transformed into E. coli BL21(DE3) to express the protein. A maximum expression of soluble protein was observed with induction by 0.1 mmol·L-1 IPTG at 37℃ for 4 hours. The fusion protein was purified through a Sepharose-Glutathione column, and verified by SDS-PAGE and Western blotting using an anti-GST polyclonal antibody. We successfully constructed the GST-AsMYC2 plasmid, produced and purified the GST-AsMYC2 fusion protein, which would provide the basic material for polyclonal antibody preparation, interactive factors screening and gene function research. According to the tissue-specific expression pattern analysis by qRT-PCR method, the AsMYC2 gene in A. sinensis tissues is mainly expressed in roots and stems, the main agarwood formation parts, and lowest expressed in leaves. These results indicate that AsMYC2 gene likely play some roles in agarwood formation in A. sinensis.

Effect of pleiotrophin on decidualization of uterine stromal cells in mice / 吉林大学学报(医学版)

Objective:To construct the pleiotrophin (PTN )overexpression vector,and to explore the effect of PTN on the decidualization of uterine stromal cells in the mice.Methods:The specific primers containing restriction enzyme cutting sites were designed according to the PTN gene sequences published in GenBank for PCR amplification.The amplified fragment of PTN was recovered from the agarose gel and cloned into the pGEM-T vector.The pGEMT-PTN was cut by double enzyme digestion and ligated into pcDNA3.1 (+)to construct the PTN overexpression plasmid.After transfection with PTN overexpression plasmid,the expression levels of PTN mRNA in the uterine stromal cells and the expression levels of decidualization markers Prl8a2 and Prl3c1 were detected by qRT-PCR method.The uterine stromal cells transfected with pcDNA3.1 (+)empty vector were used as control group. Results:The results of identification by double enzyme digestion indicated that the bands of PTN overexpression plasmid were consistent with those of the target gene,and the clone sequencing results suggested that it had 100% homology with mouse PTN gene sequence published in GenBank.Compared with control group, the expression levels of PTN,Prl8a2 and Prl3c1 mRNA in mouse uterine stromal cells in PTN overexpression group were significantly increased (P < 0.05).Conclusion:PTN overexpression could increase the expression levels of decidualization markers in mouse uterine stromal cells,indicating that PTN might play an enhancement effect during uterine decidualization in the mice.

Construction and expression of prokaryotic vector of AsJAZ1 gene from Aquilaria sinensis / 中国中药杂志

The full-length coding sequence (cds) of jasmonate-zim-domain protein (AsJAZ1) gene was cloned from Aquilaria sinensis, the prokaryotic vector was constructed and the recombinant proteins expression was induced to provide the basic material for interactive proteins screen and gene function research. In this study, with the total RNA isolated from A. sinensis leave as template, the full-length cds of AsJAZ1 gene was amplified using RT-PCR method and subcloned into pET-28a vector. The recombinant plasmid identified by restriction enzyme digestion and nucleotide sequencing was transformed into E. coli BL21(DE3). Inducing with 0.5 mmol•L⁻¹ IPTG at 37 ℃ for 4 hours, a fusion protein about 39 kDa was maximumly obtained. AsJAZ1 fusion protein had been expressed successfully mainly in the form of inclusion bodies and only a very small amount was secreted into the cytoplasm in the supernatant.

Construction of eukaryotic vector of monkey B virus glycoprotein D gene and the gD gene expression / 中国比较医学杂志

Objective To establish an eukaryotic vector of monkey B virus glycoprotein D gene and analyze the expression of gD gene in human embryonic kidney 293T cells.Method First, the protein of monkey B virus glycoprotein D was obtained by gene synthesis.The gene fragments were digested with Pst I and Not I, and ligated to pEGPF-N3. Then, the recombinant plasmid pEGPF-N3-GD was transfected into 293T cells.The expression of gD protein in the cells was detected by Western blot, and the expression localization was investigated using laser scanning confocal microscopy. Results The recombinant plasmid pEGPF-N3 carrying gD gene was successfully constructed, and normally expressed in the 293T cells.Conclusions Glycoprotein D of monkey B virus is expressed successfully in the 293T cells and the protein is located on the cell surface.It may be useful for the preparation of specific recombinant antigen to the glycoprotein D of monkey B virus on cell surface, and can be also used for preparation of antigen slide for detection of monkey B virus.

Construction of shSet7/9 vector and its function in HepG2 / 西安交通大学学报(医学版)

Objective To silence human gene Set7/9 and screen out stable transfection cell line in hepatocellular carcinoma cell line HepG2 so as to investigate the impact of down-regulation of Set7/9 in cell line HepG2 and provide experimental foundation for studies on the effect of set7/9 in HepG2.Methods The target oligo was designed and synthesized;shRNA interference vector and the control vector were constructed and transfected into HepG2 cells;the stable transfection cells were screened out.Then Real-time PCR and Western blot were performed to detect the silence of Set7/9 according to both gene expression and protein expression level. Results The shRNA interference vector was constructed and transfected into HepG2 cells successfully.Compared with that in the negative control group,the expression of Set7/9 was dramatically downregulated (P < 0.05 ). Meanwhile,the expression of related protein Sirt1 and Suv39h1 was upregulated 8.4 folds and 1.1 fold, respectively.Conclusion Downregulation of Set7/9 expression can upregulate Sirt1 and Suv39h1,suggesting that Set7/9 may affect the activity of HepG2 cell lines.

Construction of recombinant eukaryotic expression vector pCDNA3.1-MT2A and cellular localization of MT2A protein in different cell lines / 中国免疫学杂志

Objective:To construct the recombinant eukaryotic expression vector pCDNA3.1-MT2A and to investigate the cellular localization of MT2A protein in 293T and SMCC7721cell lines.Methods: Gene synthesis method was used to synthetic gene MT2A,added a Kozak sequence and His tag sequence at the N-terminus,the amplified target gene was connected to the pcDNA3.1(+) vector which was double digested between the BamH Ⅰ and Not Ⅰ.After transformation to E.coli DH5α, the positive clones were picked for plasmid extraction then Electrophoretic and sequenced.The pCDNA3.1-MT2A plasmids which passed through electrophoretic and sequenced were transfected 293T and SMMC7721 cell lines by liposome method,and then observed their expression and localization in eukaryotic cells by laser confocal microscopy.Results: The recombinant plasmid pCDNA3.1-MT2A was confirmed by restriction analysis and DNA sequencing,the sequence of the target gene MT2A was entirely correct,eukaryotic expression vector was successfully constructed and cell lines which had transfected recombinants could see the expression of green fluorescent protein in the cytoplasm.Conclusion:Successfully constructed fusion gene of pCDNA3.1-MT2A and expressed in eukaryotic cells,we found that the MT2A was mainly localized in the cytoplasm of 293T and SMMC7721 cell lines.The findings can help us to lay the foundation for the functions of MT2A in hepatoma cells.