Characterization of the functional domain of STT3a of oligosaccharyltransferase from Dunaliella salina / 生物工程学报

To investigate the function of STT3a gene in salt adaptation and flagellar regeneration of Dunaliella salina (D. salina), a pair of degenerate primers was designed according to conserved homologous amino acid sequences of VCVFTA and DVDYVL of STT3a from Chlamydomonas, Arabidopsis thaliana and other organisms. A cDNA sequence of 1 650 bp encoding a whole functional domain of STT3a was amplified from D. salina by RT-PCR and 3' Rapid Amplification of cDNA Ends (RACE), which shared homology with Chlamydomonas (48%), Arabidopsis thaliana (50%), Homo sapiens (46%), etc. Real-time fluorescence quantitative PCR (real-time Q-PCR) demonstrated that the STT3a mRNAs from D. salina were induced by increased concentration of NaCl, and increased to 11-fold higher by 3.5 mol/L NaCl than that by 1.5 mol/L NaCl (P < 0.01). Also, STT3a mRNA of D. salina maintained at a higher level in the process of flagellar regeneration with than without experiencing deflagellar treatment. In conclusion, the findings of this study demonstrate that the high expression of the STT3a gene enhances the capability of salt adaptation and flagellar regeneration in D. salina.

Construction of Dunaliella salina heterotrophic expression vectors and identification of heterotrophically transformed algal strains / 生物工程学报

We constructed inducible and constitutive heterotrophic expression vectors of Dunaliella salina (D. salina) and identified heterotrophic transformants. A gene encoding a glucose transporter (Glut1) was cloned from human placenta tissues by RT-PCR and sequenced. Inducible heterotrophic expression vector pMDDGN-Bar of D. salina, which included a duplicated carbonic anhydrase (DCA) promotor and a Bar selectable marker that could drive expression of the Glut1 gene in D. salina, was constructed by molecular biology methods. In addition, we constructed another vector G5Glut1-Bar that contained a constitutive ubiquitin promotor, Glut1 and Bar box. The two expression vectors were introduced into D. salina by electroporation method, and then screened the transformants with phosphinothricin (PPT). Total RNA of the transformants extracted was used to analyze the integration of the target gene (Glut1) by RT-PCR. The cloned Glut1 sequence was 1479 bp and encoded 493 amino acids. The results of all enzymes digesting showed that two expression vectors were successfully constructed. After screening by PPT for several weeks, the transfomants grew well whereas wild-type cells died completely. The result of RT-PCR indicated that two transformants both had an about 250 bp specific band and the sequence homology was 100% compared with the human Glut1 sequence by Blast analysis. Taken altogether, inducible and constitutive heterotrophic expression vectors of D. salina was constructed successfully and the Glut1 gene was integrated into the genome of D. salina. Expression vectors above-mentioned may be used for the expression of the foreign Glut1 gene in D. salina.

An optimal electroporation system for Dunaliella salina / 生物工程学报

To optimize the electroporation system in Dunaliella salina (D. salina), we studied the effects of growth phase of cells, electric parameters, electroporation buffer and concentration of plasmid on transformation efficiency. The results showed that a transformation efficiency of 1.81 per thousand was achieved in D. salina cells at mid-log growth phase electroporated with plasmid (DCA-bar) 10 microg/mL, voltage 0.8 kV and capacitance 25 microF. However, when glycerol was added to electroporation buffer at a final concentration of 0.4 mol/L, the transformation efficiency was increased up to 2.03 per thousand. Additionally, transformation was done with plasmids DCA-bar, NR-bar, pUomega-bar respectively, under above optimum conditions, and similar transformation efficiencies were obtained. The findings indicate that an efficient and stable system of electroporation in D. salina has been developed, providing a powerful tool for the transgenic research of D. salina.

Knockdown of NF-?B signaling pathway by siRNA inhibits the proliferation and invasiveness of HeLa229 cells / 基础医学与临床

Objective To investigate cell proliferation and invasiveness of cervical cancer HeLa229 cell after knockdown of NF-?B signaling pathway by P65 siRNA.Methods RNA interference was employed for specific inhibition of the expression of P65.HeLa229 cell was divided into transfected group and untransfected group.Cell viability was detected by MTT after the HeLa229 cells were transfected with or without P65 siRNA for 24,48,72 h.The sensitivity to 5-Fu of the HeLa229 cell,transfected with or without P65 siRNA,was evaluated also by MTT.Boyden chamber experiment in vitro was used to detect the invasion of HeLa229 cell.Results P65 siRNA inhibited the cell proliferation as compared with the untransfected cells.Proliferations of both cells transfected with and without P65 siRNA were inhibited in a concentration-dependent manner,while at the same concentration of 5-Fu the viability of transfected HeLa229 cells was significantly suppressed(P

Construction and expression of prokaryotic vector of a novel candidate gene IDD01 associated with hepatocellular carcinoma / 第三军医大学学报

Objective To construct the prokaryotic vector and to express a novel candidate gene IDD01 associated with hepatocellular carcinoma (HCC) for its functional study. Methods The open reading frame (ORF) of IDD01, amplified from HCC tissue by RT PCR, was inserted into the expression vector pMAL c2X. The recombinant vector was transformed into E. coli TB1, and the expression products were analyzed by SDS PAGE. Results The length of PCR product was about 770 bp. The restriction enzyme digestion and sequencing conformed that the gene segment was correctly inserted into the vector. SDS PAGE and density scanning indicated that the protein was expressed as a fusion protein with 28 KD of molecular weight and the fusion protein possessed 30.4% of the total bacterial protein. Conclusion The recombinant vector is constructed successfully and IDD01 can be expressed at a high level, which lays a foundation for the further research on the functions of IDD01 gene.

Construction of signal peptide-canstatin expression vector and its secretable expression in Eca-109 cells / 中国病理生理杂志

AIM: To construct signal peptide-canstatin expression vector pEGFP-C1-SP-Can and express secretable mouse canstatin fusion protein in Eca-109 cells.METHODS: Site-directed mutagenesis was used in amplifying the signal peptide of murine plasminogen to construct the plasmid pEGFP-C1-SP.The cDNA of mouse canstatin,obtained from a cloning vector pMD18T-Can by PCR,was inserted into pEGFP-C1-SP to construct a secretable expression vector pEGFP-C1-SP-Can.Constructed plasmid pEGFP-C1-SP-Can was transiently transfected into Eca-109 cells via lipofectamine,and subsequently its secretable expression in the medium of cultured Eca-109 was observed by Western blotting.RESULTS: DNA sequencing and restriction enzyme analysis attested the validity of the constructed plasmids pEGFP-C1-SP and pEGFP-C1-SP-Can.EGFPcanstatin fusion protein was proved to be secretably expressed in Eca-109 by Western blotting.CONCLUSION: It is concluded that the constructed vector pEGFP-C1-SP-Can is valid and capable of expression in Eca-109,these findings provide a basis for testing the function of mouse canstatin and its application in gene therapy.

Cloning and expression of mouse canstatin cDNA in E.coli / 中国病理生理杂志

AIM: To clone and express mouse canstatin (m canstatin)cDNA and provide a basis for the further research on its anti-angiogenic activity and potential application for cancer therapy. METHODS: Total RNA was extracted from mouse liver tissue by Trizol Reagent, and mouse canstatin cDNA was amplified by RT- PCR, then cloned into vector pMD18-T for sequencing. pET30a(+)-m canstatin recombinant plasmid was constructed and expressed in E.coli BL21 with induction of IPTG. RESULTS: Mouse canstatin cDNA is 684 bp coding 227 amino acids. The sequences of both cDNA and amino acid share high homology with human canstatin, with cDNA identity at 89% and amino acids identity at 96% to human canstatin. In the present study, pET30a(+)-m canstatin recombinant plasmid was expressed in E.coli BL21. CONCLUSION: Mouse canstatin cDNA has been cloned for the first time. Constructed pET30a(+)-m canstatin recombinant plasmid is highly expressed in E.coli BL21.