Abstract: Previous studies have revealed Twist, a basic helix-loop-helix transcription factor, plays an important role in breast cancer metastasis. To clarify the molecular mechanism of its involvement in cancer metastasis, Twist was silenced by RNAi in highly metastatic 4T1 cells. Then microarray chips were used to investigate the gene-expression pattern of the Twist-knockdown 4T1 cells and the normal 4T1 cells. The results indicated that silencing of Twist significantly suppressesed lung metastasis of 4T1 cells in vivo. Direct comparison of gene-expression profiles showed that 167 genes in Twist-knockdown cells differed dramatically in expression levels from those in control cells. Among the 167 genes, we further found 26 well-known tumor-associated genes, including 15 up-regulated and 11 down-regulated genes. These genes appear to be regulated by Twist during breast tumorigenesis. The findings of this study provide new insights into the mechanism by which Twist is involved in tumorigenesis.
Objective:To construct NBS1 microRNA expressing eukaryotic recombinants, and identify biological activity of recombinants in Hela cell after transfection . Methods: According to sequence of NBS1mRNA, the NBS1 pre-microRNA was designed and synthesized, then cloned into the GFP reporter pcDNA6.2-GW/EmGFP-miR vector and transfected into Hela cell line. To detect integrity of inset fragment through colony PCR and sequencing analysis. To identify interference efficiency of NBS1 miRNA recombinants by way of Real-Time PCR 12h after thansfection and determine biological activity of recombinants . Results: Sequences of inset fragment in four microRNA expressing recombinants were correct . NBS1 mRNA expression of four microRNA recombinants were 0.24±0.17 (NBS1mi-1 recombinant), 0.12±0.12 (NBS1mi-2 recombinant), 0.41±0.97 (NBS1mi-3 recombinant), 0.48±0.93 (NBS1mi-4 recombinant),that is the lowest in the NBS1mi-2 group.Conclusion: Four NBS1-targeting microRNA expressing recombinants all have biological activity in Hela cell line, and NBS1mi-2 recombinant has the most interference efficiency. The microRNA expressing plasmidwhich were successfully constructed and lay foundation for the studies on the tumor gene therapy of microrna targeting NBS1.
ecombinant interferon alpha (IFN-alpha) has been proven useful for treating a variety of human cancers and viral diseases. Because of its short circulating half-life IFN-alpha is administered to patients by daily or thrice weekly injection for optimal effectiveness. Recombinant consensus interferon mutant Ⅱ (IFN-Con-m2, IIFNm2) was a mutant from IFN-Con with a free cysteine residues at position 86.The protein could be modified with a 2 kDa-maleimide PEG and the mono-PEGylated proteins were purified by CM Sepharose Fast Flow column chromatography. Mono-PEGylated IIFNm2 could be separated from multi-PEGylated IIFNm2 and unmodified IIFNm2 by stepwise elution. After purification, the purity of mono-PEG-IIFNm2 was up to 98%, and the biological activity was more than 5.0×106IU/mg. The test of anti-trypsin digestion suggested that PEGylation could improve the ability of avoiding the trypsin digestion. Pharmacokinetic experiments in rats demonstrated that the circulating half-life of the PEGylated protein was up to 11.5-fold longer than that of IIFNm2. These data could demonstrate the utility of site-specific PEGylation for creating highly potent, long-acting IIFNm2.
Humanin (HN, its analogue [Gly14]-Humanin, HNG) was originally identified as an endogenous peptide that protects neuronal cells from apoptosis induced by various types of Alzheimer's disease-related insults. But the relative low content of this peptide in its natural sources limits its further characterization. In this study, we constructed an expression vector pET32a/HNG and transformed it into E. coli BL21 trxB (DE3). HNG was expressed as a fusion protein in the soluble fraction and was purified by nickel affinity chromatography. Subsequently, the purified fusion protein was cleaved by enterokinase and was further purified by reverse-phase HPLC. We purified 23 mg recombinant HNG (rHNG) from 1 L bacterial culture. The molecular weight of rHNG determined by ESI-MS was 2876.5 Da which was the expected size for correctly processed peptide. The N-terminal amino acid sequence of rHNG determined by Edman degradation method is identical to the theoretical sequence. Neuroprotective bioassay studies of rHNG exhibited its potential neuroprotective effect comparable to that of the natural HNG peptide.
An eukaryotic expression plasmids pEGFP-C1-A3I3,with open reading frame(ORF) of FMDV WFL strain, was constructed based on the pEGFP-C1.The results of transient expression showed that the eukaryotic expression plasmids can be expressed in BHK-21.When cotransfected the plasmid with RNA transcribed from linearized cDNA clone,FMDV antigen and nucleotide acids was detected in cell culture fluid by sandwich ELISA and RT-PCR,respectively.And the virus particles were also found by electron microscope.The results proved that cotransfection of eukaryotic expression plasmid with RNA can improve virus rescuing.And for the first time we successfully rescued FMDV by cotransfecting eukaryotic expression plasmid with RNA transcribed in vitro.
PhoQ gene that was amplified by polymerase chain reaction(PCR)from Salmonella typhimurium genome DNA was cloned into a procaryotic expression plasmid pUC18, sequenced and GenBank accession No. is DQ787014. The identified recombinant plasmid was then used to transform into the competent Salmonella, and the positive clones were screened by PCR and restriction enzyme digestion.and phoQ recombinant protein was expressed by IPTG induced method. PhoQ gene expression was analyzed by SDS-PAGE. The stability of recombinant bacterial cultured in vivo was observed. It showed t hat recombinant bacterium could stably reproduction, growth and hand down from generation to generation in vivo. To investigate the effects of different virulence of the recombinant S. typhimurium and wildness S. typhimurium, which were tested in 45 days old asepsis' KM mice by nonnasality rejection. The data showed that the LD50 of the recombinant and wildness strains were 3.981×107 cf u/ mL and 5.012×102 cf u/ mL ,respectively. It suggests that the different virulence between the two strains were occurred, the wildness S. typhimurium was more virulent than the recombinant S. typhimurium. The result testified phoQ gene is one of the important regalatots in the virulence mechanism of S. typhimurium.
To study the role of hCD46 in mediating the specific adhesion by N. gonorrhoeae to its human host, an hCD46 minigene including the promoter and cDNA was synthesized by using ligation-mediated PCR, and cloned into vector pcDNA3.1 replacing the original CMV promoter. The resultant recombinant vector pCD46 was transfected into COS-1 cells. The effective expression of hCD46 on the membrane of COS-1 cells was detected by indrect immunofluorscence assay; Western blot revealed that the molecular weight of the recombinant hCD46 was correct. The hCD46 expressing COS-1-46 cells were sorted by FACS and could be adhered by piliated N. gonorrhoeae stain YZ1228 revealed by bacteria binding assay. These data indicate that hCD46 is a kind of crucial pilus receptor that mediates the host specific adhesion by N. gonorrhoeae, and the hCD46 minigene, pCD46, can be used to develop transgenic mice model for the study of gonococcal diseases.
To express and purify recombinant mature adhesive protein （rCPM36）of avian Pasteurella multocida type strain P1059 and detect its antigenicity. Methods: The cpm36 gene, encoding a mature adhesive protein without signal peptide was amplified by PCR from genomic DNA of avian Pasteurella multocida strain P1059, PCR product was cloned into the expression vector pQE30 to constructed recombinant plasmid pQE30-cpm36 and then transformed into E. coli M15, inducing it with 0.2mmol/L IPTG for 4 hours. The expression and solubility of recombinant protein was detected by DSD-PAGE. The interest protein was purified by Ni-NTA affinity chromatography and its antigenicity was detected Western blot analysis. Results: SDS-PAGE showed the 37kDa recombinant protein was successfully expressed in E.coli after IPTG inducing. In Western blot analysis，rabbit antiserum against rCPM36 reacted with rCPM36, it demonstrated that the recombinant protein is an antigenic protein. Conclusion: rCPM36 is successfully expressed in E. coli. The interest protein is successfully purified by Ni-NTA affinity chromatography and is detected its antigenicity. The rCPM36 protein might be a useful vaccine candidate antigen for avian Pasteurella multocida.
To clone and analyze the 5’ upstream region of the small subunit (rbcS) of ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco), The genomic DNAs from Dunaliella salina were digested with Dra I, EcoR V, Pvu II and Stu I, respectively. GenomeWalker Adaptors were then ligated to the ends of the digested DNA fragments. Accordingly, GenomeWalker Libraries including GWL 1, GWL 2, GWL 3 and GWL 4 were constructed. The 5’ upstream regions of rbcS were amplified from the above 4 GenomeWalker Libraries by nested PCR. Single major PCR product of about 1.2 kb from the GWL 1 and GWL 4 was generated. The partial sequences of 3’-end in the 1.2 kb fragment from GWL 4 was completely consistent with the sequences of 5’-end of the rbcS cDNA, indicating that the fragment was located the upstream of the rbcS cDNA. Several conserved promoter motifs, such as TATA-like box, CAAT-like box, etc, and the tandem GT were found in the fragment. The sequences 0f about 800 bp located downstream of the EcoR I site in the 1.2 kb fragment was fused with bar-nos polyA to generate the expression vector pSP-B. pSP-B was trandformed into the cells of D. salina by electroporation. PPT-resistant phenotype tranaformants were isolated, The results of PCR and Southern bolts of the transformed D. salina showed bar gene had been integrated into the genome of D. salina. It is concluded that the sequence we isolated from D. salina was the 5’ upstream region of the rbcS gene and possessing activity of promoter, thus it can be used in the construction of the bio-reactor of transgenic D. salina.
Using the character of natural aggregation of CHO cells, and an ultrasonic and sedimentation column combined perfusion system to promote cells aggregation and retention into bioreactor, we cultured successfully recombinant CHO cell strain MK3-A2, which could secrete TNK-tPA, by a serum-free perfusion culture system. The culture periods in our two experiments were as long as 77 and 110 days respectively. The cells density reached 2×107 cells /ml. The average volumetric productivity of TNK-tPA was 89 mg/L.d, and the highest one was 216mg/L.d.The cells aggregation rate was approximately 90%, and the diameters of most of them were 285～570μm. During the perfusion culture the cells retention rate almost kept in 95% and the viability of cells was more than 85%.Thus, it means that aggregation culture with such perfusion system could be used to scale up produce biopharmaceuticals instead of microcarrier culture system.
Using the strain of Rhizopus oryzae ME-F12, the fermentation conditions for fumaric acid were studied for the purpose of optimizing both fumaric acid production and productivity. An orthogonal experiment was designed to investigate the influence of glucose concentration, urea concentration, seed age and inoculation size. The optimal seed age and inoculation size was determined as 36h and 6ml, respectively. A Desirability function based on response surface analysis was further applied to perform the multiple variable optimization. The best glucose and urea concentration were determined as 132.73g/l and 0.0586g/l, respectively. The predicted value for fumaric acid production, productivity and the Desirability value were 71.42g/l, 0.804g/(l.h) and 0.966, respectively. Verification experiment was carried out on a 5L fermentor. After a 88h fermentation period, 130g/l glucose was converted into 66.5g/l fumaric acid with the productivity of 0.755 g/(l.h). Comparing to the value before optimization, the production and productivity of fumaric acid were increased by 13.9% and 15.8%. Results indicated that both the production and productivity were successfully optimized simultaneously, which laid a solid foundation for the further scale-up.
The specific fragment of Pneumococcal surface protein A( PspA) and Pneumococcal Surface Adhesin A(PsaA) gene was amplified by PCR from Streptococcus pneumonia 5. The amplified fragnent of PspA and PsaA gene was ligated into pET- 27b (+) vector and transformed into BL 21 E.coli for expression and obtain the expressive production of PspA and PsaA. Induced by IPTG, the expression level was as high as 75 % of total protein. The result showed that the recombinant plasmid could express a specific 75 kDa and 37 kDa fusion protein in E. coli BL 21, which showed the good immunogenicity and a broadly cross reactivity with the other serotypes.
Objective: Clone, express and purify human recombinant calreticulin (CRT). Methods: Human CRT cDNA was amplified by RT-PCR from total RNA of human lung cancer cell line A549 cells. Then, PCR product was subcloned into prokaryotic expression vector pET-15b. After sequencing, this recombinant plasmid was transformed into E. coli. Rossetta. Recombinant CRT was expressed in host cells by IPTG induction. Resulted protein was purified by Ni-NTA resin under denature condition and dialyzed to recover its native structure. SDS-PAGE and Western blot method were used to identify the expression and purification of reconbinant CRT. Results: Human CRT cDNA was cloned from total RNA of A549 cells. CRT prokaryotic expression vector pET-15b-crt was constructed. Reconbinant CRT was induced to express in E. coli and purified by Ni-NTA affinity chromatograph. Conclusion: A method for prokaryotic expression and purification of human recombinant CRT was successfully established. This method laid a foundation for the succedent CRT research.
RT-PCR amplification of ginseng β-amyrin synthase gene was successfully performed based on the total RNAs extracted from ginseng hairy roots induced by Agrobacterium rhizogenes A4 using a modified guanidine isothiocyanate-method. Sequence analysis of this gene revealed that its sequence was consistent with the sequence of a previously reported ginseng β-amyrin synthase gene (GeneBank No. AB009030). This gene was inserted into pMD-119T simple vector and transformed into E. coli DH5α. Furthermore antisense plant expression vector of this gene was constructed using the pBI121 vector, laying foundation for studies on antisense regulation of ginseng β-amyrin synthase gene.
Objective: To investigate the biotransformation of eight active constituents chemicals by hairy roots of Polygonum multiflorum Thunb. Methods: Active compounds were added to the culture system of hairy roots of P. multiflorum after precultured for 9 d, then they were co-cultured for another 7 d. The products were isolated and purified by column chromatography. Results: Three of the eight active constituents were identified to be biotransformed, which were furannoligularenone (6), 1,4-benzenediol (7) and artemisinin (8). HPLC analysis of the biotransformation of 6 showed two new peaks except the peak of substrate, then the corresponding two compounds were obtained; Substrate 7 was converted into arbutin. In the case of 8, there were two new peaks and the corresponding two monomers have been also isolated. Conclusion: The result showed that there were several enzymes in the culture system of P. multiflorum hairy roots, which could processed glycosylation, oxidation, reduction and hydroxylation reactions.
Cationic antimicrobial peptides are produced by all organisms, from plants and insects to human beings, as a major part of their immediately effective, nonspecific defences against infections. Although many demonstrate direct antimicrobial activity against bacteria, fungi, eukaryotic parasites and/or viruses, it has been established that antimicrobial peptides have a key modulatory role in the interface of innate and adaptive immunity. More recent evidence suggests that antimicrobial peptides are effective adjuvants, are synergistic with other immune effectors, initiate the adaptive response, support wound healing, induce or modulate of chemokine and cytokine production, alterate of gene expression in host cells, and inhibit of proinflammatory responses of host cells.In addition, the mechanisms of action are being unraveled, which support more effective implementation of derivatives of these endogenous peptides as therapeutic agents in overcoming infectious diseases.
Abstract: Quorum sensing is a cell-density-dependent regulatory mechanism.A lot of bacteria have the ability to secrete one or more kinds of signal molecules.They utilize the signal molecules to feel the bacteria density and regulate a series of target genes expression,but the effect of signal molecules on the production of antibiotics is the investigation focus.This article reviews the new advancement of quorum sensing mechanism,the effect of it on the antibiotics production,and pay more attention to Burkholderia cepacia.
Bacillus megaterium is a potential kind of genetic engineering host which can secrete, Now it is used in industry and academic fields. It has some advantages, e.g: protein yields are exceptionally high; heredity is very stable; production can be secreted; zymotechnique is mature and so on. This article has summarized the advantages of Bacillus megaterium ,the characteristics of the expression vector, the methods of transformation of Bacillus megaterium,the expression of foreign proteins, and the strategies of high-level expression, meanwhile looked into the future of the Bacillus megaterium.
Hyaluronic acid (HA) is a linear polysaccharide chain composed of alternating β-1,4-glucuronic acid (GlcA) and β-1,3-N-acetylglucosamine (GlcNAc) moieties. Construction of engineering strain has become the prevailing strategy for increasing yield and improving its quality, especially the molecular weight. Here the molecular mechanism of HA biosynthesis in Streptococcus strains was reviewed, involving fermentation strains, operon structure, crucial enzyme and construction of engineering strains. In addition, the prevalent problems in HA fermentation production were also discussed and the protocols were tentatively put forward for the upcoming research and industrial production.
Environmental pollution is a growing global problem. Conventional approaches for clean-up of contaminated sites are not only costly and low efficient but also cause other damage to the environment. Phytoremediation has gained more importance in recent times as an alternative technology for environmental restoration. In this review, five types of phytoremediation strategies including phytoextraction, phytodegradation, phytovolatilization, phytofiltration, and phytostabalization are summarized. In addition, recent progresses in development of transgenic plants with enhanced potential for remediation are reviewed.
