Construction of T7 RNA polymerase gene expression system in Anabaena sp. PCC 7120 for the expression of hG-CSF / 生物工程学报

The low expression rate of exogenous genes in cyanobacteria is one of the bottlenecks of cyanobacteria genetic engineering. The T7 RNA polymerase expression system has achieved the efficient expression of exogenous genes in Escherichia coli. Cyanobacteria and E. coli are both Gram-negative bacteria with high genetic homology. The construction of T7 RNA polymerase expression system in cyanobacteria may improve the expression of foreign genes. In order to construct the T7 RNA polymerase expression system in Anabaena sp. PCC 7120, methods such as overlapping extension PCR and digestion-ligation technique were used to construct a site-specific integration vector pEASY-T1-F1-TacT7RNAPCmR-F2 and a shuttle expression vector pRL-T7-hG-CSF. The site-specific integration vector is capable of expressing T7 RNA polymerase, and the shuttle expression vector expresses hG-CSF driven by the T7 promoter. Then we introduced the site-specific integration vector into the wild type cyanobacteria by electroporation and transferred the shuttle expression vector into the site-integrated transgenic cyanobacteria by triparental conjugative transfer. In the end, we identified the presence of foreign genes in cyanobacteria by PCR, tested the transcription level of foreign genes in cyanobacteria by RT-PCR, and detected the protein expression of foreign genes in cyanobacteria by Western blotting. The two vectors were successfully constructed, the T7 RNA polymerase gene and hG-CSF gene were transferred into cyanobacteria well, and both genes were also expressed in cyanobacteria. In summary, the T7 RNA polymerase expression system was successfully constructed in cyanobacteria, and the expression rate of hG-CSF gene was doubled than the traditional cyanobacteria expression systems. This expression system will provide a better tool for the application of cyanobacteria genetic engineering and will promote the development of cyanobacteria as a chassis cell in the fields of synthetic biology in the future.

Identification of protein that interacts with adhesion protein of Mycoplasma genitalium from T7-phage display cDNA library / 中国免疫学杂志

Objective:To screen and identify the protein that interacts with the adhesion protein of Mycoplasma genitalium (MgPa)from T7-phage display cDNA library of human uroepithelial cells(SV-HUC-1).Methods:Recombinant adhesion protein of My-coplasma genitalium(rMgPa)was used as target molecule to biopan the T7 phage display cDNA library of SV-HUC-1 cell,the selected positive clones were analysed using DNA sequencing and BLAST analysis and identified by means of indirect ELISA,Dot immunoblot and Far-western blot.Results:After four rounds of biopanning,positive phages were obviously enriched.According to the results of DNA sequencing and BLAST analysis,the selected randomly 32 positive clones included 7 kinds different sequences,of which the number of RPL35 repeats was the most.The results of indirect ELISA,Dot immunoblot and Far-western blot showed that 7 representative phages could bind specifically with rMgPa.Conclusion:60S ribosomal protein L35(RPL35) may be the interacting protein of MgPa,which lays the experimental foundation for understanding the function of MgPa and the pathogenesis of Mycoplasma genitalium.

Development of a novel vector for cloning and expressing extremely toxic genes in Escherichia coli

Background: Escherichia coli has been widely used as a host to clone and express heterologous genes. However, there are few vectors available for cloning and expressing extremely toxic genes, which limits further basic and applied research on extremely toxic proteins. Results: In this study, a novel vector pAU10 was constructed in E. coli. pAU10 utilizes the combination of the efficient but highly repressible T7-lacO promoter/operator and the strong rrnBT2 transcriptional terminator upstream of the T7 promoter to strictly control unwanted transcription of the extremely toxic gene; in addition, the trp promoter/operator is oriented opposite to the T7 promoter to control the production of the antisense RNA that may block the translation of leaky mRNA. Without the supplementation of IPTG and L-tryptophan in the culture medium, transcription of the extremely toxic gene by the T7 promoter is highly repressed, and the trp promoter produces the antisense RNA, which strictly prevents unwanted expression of the extremely toxic protein in E. coli. With the supplementation of IPTG and L-tryptophan, the T7 promoter efficiently transcribes the extremely toxic gene, and the trp promoter does not produce the antisense RNA, ensuring efficient expression of the extremely toxic protein in E. coli. Tight regulation and efficiency of expression of an extremely toxic gene cloned in the vector pAU10 were confirmed by cloning and expressing the restriction endonuclease-encoding gene bamHI without its corresponding methylase gene in E. coli JM109(DE3). Conclusion: pAU10 is a good vector used for cloning and expressing extremely toxic genes in E. coli.

Molecular cloning, expression, and immobilization of glutamate decarboxylase from Lactobacillus fermentum YS2

Background: GABA (γ-aminobutyric acid) is a four-carbon nonprotein amino acid that has hypotensive, diuretic, and tranquilizing properties. Glutamate decarboxylase (GAD) is the key enzyme to generate GABA. A simple and economical method of preparing and immobilizing GAD would be helpful for GABA production. In this study, the GAD from Lactobacillus fermentum YS2 was expressed under the control of a stress-inducible promoter and was purified and immobilized in a fusion form, and its reusability was investigated. Results: The fusion protein CBM-GAD was expressed in Escherichia coli DH5α carrying pCROCB-gadB, which contained promoter PrpoS, cbm3 (family 3 carbohydrate-binding module from Clostridium thermocellum) coding sequence, the gadB gene from L. fermentum YS2 coding for GAD, and the T7 terminator. After a one-step purification of CBM-GAD using regenerated amorphous cellulose (RAC) as an adsorbent, SDS-PAGE analysis revealed a clear band of 71 kDa; the specific activity of the purified fusion protein CBM-GAD reached 83.6 ± 0.7 U·mg-1. After adsorption onto RAC, the immobilized GAD with CBM3 tag was repeatedly used for GABA synthesis. The protein-binding capacity of RAC was 174 ± 8 mg·g-1. The immobilized CBM-GAD could repeatedly catalyze GABA synthesis, and 8% of the initial activities was retained after 10 uses. We tested the conversion of monosodium glutamate to GABA by the immobilized enzyme; the yield reached 5.15 g/L and the productivity reached 3.09 g/L·h. Conclusions: RAC could be used as an adsorbent in one-step purification and immobilization of CBM-GAD, and the immobilized enzyme could be repeatedly used to catalyze the conversion of glutamate to GABA.

Experimental study on anti-angiogenic activity of T7 peptide derived from active fragment of Tumstatin / 中国现代普通外科进展

Objective:To investigate the influence and related mechanisms of T7 peptide derived from tumstatin on angiogenesis in vitro.Methods:HUVECs were incubated in hypoxia chamber(37℃,1％ O2,5％ CO2,94％ N2) to simulate the hypoxic microenvironment in tumors,and grouped into:hypoxia group,hypoxia+T7 peptide (1.0 μ mol/L) group,and hypoxia+T7 peptide(2.0μ mol/L) group.The tube formation assay was applied to analyze the influence of T7 peptide on angiogenesis under hypoxia.Cell Counting Kit-8 was applied to observe the cell viability.The apoptosis rate was detected with Annexin V-FITC,and the expression levels of pro-apoptotic protein BAX and anti-apoptotic protein Bcl-2 were observed with western blot.Immunofluorescence and western blot were used to detect the expression of VE-cadherin.Results:Under hypoxic condition,T7 peptide significantly inhibited capillary-like tube formation (P＜0.05),inhibited ECs viability(P＜0.05),and increased the ECs apoptosis rate in vitro;T7 peptide resulted in the downregulation of anti-apoptotic protein Bcl-2 (P＜0.01)and upregulation of pro-apoptotic protein BAX(P＜0.05);the expression of VE-cadherin was downregulated by T7 peptide significantly(P＜0.05).Conclusions:T7 peptide could execute its anti-angiogenic activity via inhibition of ECs viability,induction of ECs apoptosis rate,and downregulation of VE-cadherin expression.

Screening protein interacted with enterovirus 71 3A protein by means of T7-phage display system / 国际检验医学杂志

Objective To screen and identify protein interacted with enterovirus 71 (EV71)3A protein by means of T7-phage display system.Methods The prokaryotic expression vector of 3A protein was constructed for expressing and purifying 3A pro-tein.With 3A protein as the target protein,the T7-phage display system was adopted to screen the human hepatocyte cDNA library. The screened products were performed the DNA sequence analysis and the homologous research.Results 3A protein was expressed and purified,after 4 rounds of biopanning,37 positive clones were selected.The inserted fragments were amplified by PCR using specific T7 primer.The products were sequenced,2 proteins were identified to interact with 3A protein by homologous analysis. Conclusion Trough the T7 phage display system,two proteins interacted with 3A are obtained.The possible function of 3A protein can be speculated by researching the function of these two proteins,which lays the foundation for the further studying the pathogen-ic mechanism of EV71.

Liquid based formulations of bacteriophages for the management of waterborne bacterial pathogens in water microcosms.

Water resources are contaminated by life-threatening multidrug resistant pathogenic bacteria. Unfortunately, these pathogenic bacteria do not respond to the traditional water purification methods. Therefore, there is a need of environmentally friendly strategies to overcome the problems associated with the antimicrobial resistant bacterial pathogens. In the present study, highly potent lytic phages against multidrug-resistant Salmonella enterica serovar Paratyphi B, Pseudomonas aeruginosa and Klebsiella pneumoniae were isolated from the Pavana river water. They belonged to the Podoviridae and Siphoviridae families. These phages were purified and enriched in the laboratory. Monovalent formulations of φSPB, BVPaP-3 and KPP phages were prepared in three different liquids viz., phage broth, saline and distilled water. The phages were stable for almost 8-10 months in the phage broth at 4 °C. The stability of the phages in saline and distilled water was 5-6 months at 4 °C. All of the phages were stable only for 4-6 months in the phage broth at 30 °C. The monovalent phage formulation of φSPB was applied at MOI < 1, as disinfectant against an exponential and stationary phase cells of Salmonella enterica serovar Paratyphi B in various water microcosms. The results indicated that there was almost 80 % reduction in the log phase cells of Salmonella serovar Paratyphi B in 24 h. In stationary phase cells, the reduction was comparatively less within same period. At the same time, there was concomitant increase in the phage population by 80% in all the microcosms indicating that φSPB phage is highly potent in killing pathogen in water. Results strongly support that the formulation of φSPB in the phage broth in monovalent form could be used as an effective biological disinfectant for preventing transmission of water- borne bacterial pathogens, including antimicrobial resistant ones.

A Novel Translocation Involving RUNX1 and HOXA Gene Clusters in a Case of Acute Myeloid Leukemia with t(7;21)(p15;q22)

Translocations involving chromosome 21q22 are frequently observed in hematologic malignancies including acute myeloid leukemia (AML), most of which have been known to be involved in malignant transformation through transcriptional dysregulation of Runt-related transcription factor 1 (RUNX1) target genes. Nineteen RUNX1 translocational partner genes, at least, have been identified, but not Homeobox A (HOXA) genes so far. We report a novel translocation of RUNX1 into the HOXA gene cluster in a 57-year-old female AML patient who had been diagnosed with myelofibrosis 39 months ahead. G-banding showed 46,XX,t(7;21)(p15;q22). The involvement of RUNX1 and HOXA genes was confirmed by fluorescence in situ hybridization.

A phage display combined with DNA affinity magnetic system can be applied to a screening of DNA binding proteins, such as transcription factors / Un fago pantalla combinado con un sistema de afinidad magnético al ADN se puede aplicar al exámen de las proteínas de unión al ADN, tales como los factores de transcripción

Here we introduce a new approach for the screening of DNA binding proteins, using a phage library based on a phage display technique. In principal, a complementary DNA (cDNA) library based on the recombinant bacteriophage T7 expressing target proteins on its capsid (phage display) is constructed. These phage particles are hybridized with a biotinylated target DNA fragment which is immobilized on the surface of streptavidin paramagnetic particle (SA-PMP). The phage particles are released from the target DNA fragment by a nuclease treatment and the recovered phages are used to the next round of hybridization. These processes are repeated three times to amplify the target phages in the population. This simple method is faster, and more systemic than other current methods (e.g. yeast one hybrid system). As a proof of this principle, we tried to isolate transcription factors which specifically bind to the promoter region of the Arabidopsis thaliana AtGST11 gene. Two obtained candidates, RING zinc finger protein and AtHB6, showed DNA binding activity to the AtGST11 promoter region. We could validate that our new application of phage display is a superior method for isolation of DNA binding proteins with a broad range of potential applications.

Large Protein Production Through Split Intein-Mediated Trans-Splicing / 中国生物工程杂志

Npu DnaE intein was used to produce some large proteins,which were difficult to obtain through conventional expression systems.A T7 expression system was described,by which the gene of T7 RNA polymerase is split into two pieces,and each piece fuses with Npu DnaE N-and C-terminal sequences respectively.Functional T7 RNA polymerase is created by mixing the two kinds of fusion constructs in vitro.The approach of split intein-mediated production of large proteins,in theory,readily generalizable to the purification of other large,cytotoxic or membrane proteins.

Establishment of IBRS-2 Cell Line Stably Expressing T7 RNA Polymerase and Recovery of SVDV From IBRST7 Cells / 生物化学与生物物理进展

The bacteriophage T7 RNAP gene was amplified via PCR from -lysogen DE3, and the gene was cloned into pBABEpuro retrovial vector, a recombinant plasmid named as pT7BABEpuro was constructed and sequenced. Then the pT7BABEpuro and pVSV-G plasmids were cotransfected into GP2-293 packaging cells by liposomese, some pseudotype viruses were ingathered and transfected into IBRS-2 cell under polybrene. The IBRS-2 cell was propagated in DMEM with puromycin. The genome extraction from the cells transfected different times, the T7 RNAP gene was amplified from the genome by PCR, the mRNA of T7 RNAP protein expressed in IBRST7 cells was analyzed by RT-PCR, respectively, the results showed the T7 RNAP gene had been integrated into the chromosome of IBRS-2 cell and expressed stably at high level. To study whether T7 RNAP is of transcriptional activity in the established IBRST7 cell line, a plasmid pIERS-EGFP-ET with a reporter gene (EGFP) under control of the T7 promoter was constructed. IRES element from FMDV (for CAP-independent translation) was cloned into plasmid pET-43.1a-c(+) downstream of the T7 promoter sequence, then EGFP gene was cloned in frame downstream of the AUG codon of the FMDV IRES, resulting in the plasmid. IBRST7 cells were transfected with plasmid pIERS-EGFP-ET using lipfection, EGFP was expressed, the results showed the T7 RNAP in IBRST7 cells has transcriptional activity. IBRST7 cell line was directly transfected with linearized full-length cDNA of swine vesicular disease virus (SVDV) HK/70, infectious SVDV was efficiently recovered from the cDNA. The reverse genetic procedure is simplified to a faster, one step protocol to recover RNA virus and will be useful to understand the mechanisms of molecular pathology of RNA virus and develop effective vaccines.

Molecular Analysis of Salivary Gland Branching Morphogenesis / Oral Science International

Recently, clinicians and scientists have focused on tissue engineering for regenerative medical therapy. This approach promises to provide remarkable clinical breakthroughs for the future. In oral and craniofacial medicine, most scientific approaches to tissue engineering currently involve tooth and bone, while little progress has been made toward regenerating organs such as salivary gland. To develop strategies for salivary gland regeneration, it will be important to understand the molecular mechanisms of normal salivary development. This mini-review describes a recently developed and tested set of approaches for identifying and characterizing molecules essential for branching morphogenesis and other developmental processes. It shows the value of using laser microdissection and the new process of T7-SAGE for gene discovery of putative candidate molecules that may be crucial regulators or mediators. We describe a stepwise series of associated strategies for reliable identification and functional testing of a candidate molecule, as well as its successful application to a specific candidate molecule originally identified by T7-SAGE.

Screening of proteins interacting with hepatitis C virus core protein from T7-phage display library / 第三军医大学学报

Objective To screen and identify proteins that interact with the hepatitis C virus core protein by means of T7-phage display system. Methods The hepatitis C virus core protein was expressed by prokaryotic expression and used as selected molecule to biopan the T7 human liver cDNA library. The selected positive clones were identified by DNA sequence and analyzed with BLAST program in GenBank. Results After BLAST in all positive clones, one protein--Smad interacting protein 1 (SIP1) was found to interact with the hepatitis C virus core protein. Conclusion T7-phage display system is a convenient, rapid and effective method for screening interacting proteins.

A Case of Greig Cephalopolysyndactyly Syndrome

Greig cephalopolysyndactyly syndrome (GCPS) is a disorder characterized by postaxial polydactyly of the hand, broad or occasionally bifid thumbs, preaxial polydactyly of the feet, broad halluces, syndactyly of the fingers or toes, macrocephaly, frontal bossing, hypertelorism and a broad nasal bridge. Intelligence is usually normal, although borderline IQ has been reported. Advanced bone age, mild hydrocephalus, craniosynostosis and agenesis of the corpus callosum are occasionally associated abnormalities. We report here a 10-day-old male infant with GCPS. Birth Weight was 2,400kg and gestational age was 39 wks. He had a wide broad high forehead, hypertelorism, broad nose base and cryptorchidism. He had preaxial polysyndactyly due to duplication of the right thumb and left accessory thumb, duplication of both halluces and syndactyly of both toes and fingers. His brain MRI showed corpus callosum agenesis, mild hydrocephalus and small choroid plexus cyst. High resolution chromosomal analysis showed a de novo balanced translocation 46, XY, t (7;8) (p22;q24.1). We report the first GCPS case in Korea with brief literature.

Inhibition of VEGF gene expression in Hela cell by short interfering RNAs / 中国免疫学杂志

Objective:To investigate the interfering effect of siRNA on the expression of VEGF in Hela cells.Methods:Three pairs of siRNA were designed according to the 1-5 extrons sequence of VEGF and synthesized by T7 RNA ploymerase transcription in vitro. To evaluate the inhibition activity of siRNA, Hela cells were transfected via siPORT Lipid. The interfering effect of siRNAs in hRPE cells was examined by semi-quantitative RT-PCR and immunofluorescence technique.Results:The results showed that the three pairs of siRNA could effectively inhibit gene expression of VEGF in Hela cells with the rates of 88.7%, 94.2% and 80.3%. But the 1-base mismatched siRNA and ssRNA(+) didn′t show significant interfering effect. Furthermore, it was found that siRNAs had a dose dependent inhibitory effect(5-10 pmol).Conclusion:The siRNAs synthesized by T7 RNA ploymerase in vitro could effectively and specifically interfere the expression of VEGF in Hela cells, providing a novel approach for gene therapy of tumor.

A Case of Split Hand Split Foot Anomaly

We experienced a case of split hand split foot(SHSF) anomaly in a male neonate who had a deficiency of the middle finger, syndactyly of the 1st and 2nd finger and a deep median cleft in both hands. He also had a deep median cleft between 2nd and 3rd toe, syndactyly of the 1st and 2nd, 3rd and 4th toe without deficiency of the middle toe in both feet. SHSF anomaly may occur either isolated or associated with other anomalies. In this case, it occurd sporadically without family history and showed an isolated type without any other specific anomalies except both posterior iris synechiae. The karyotype of patient showed 46,XY,t(7p:14q) which has not been reported yet. We reported the case with the review of the associated literatures.

A simple protocol of DNA sequencing with 10% formamide for dissolving G/C compression

Formamide has been widely used in urea/polyacrylamide gel to solve the compression problems that are occasionally found during the DNA sequencing of G/C rich regions. In this study, however, 10% formamide was added in annealing solution in stead of adding to the gel. The compressions were unfolded efficiently with a more rapid annealing reaction on ice in the presence of 10% formamide.

Molecular cloning and sequencing of mcrA locus and identification of McrA protein in Escherichia coli.

The Mcr systems (previously known as Rgl systems) of Escherichia coli recognize and cleave specific sequences carrying methylated or hydroxymethylated cytosines. We have cloned the mcrA gene and determined its nucleotide sequence. An 831 base pair sequence encodes the McrA protein. Analysis of the sequence data reveals that there arc additional ORFs internal to the above. A phage T7 expression system was used to determine the protein products encoded by the cloned mcrA gene. The results clearly show that a 31 kDa polypeptide is responsible for McrA activity. This is in agreement with the molecular weight deduced from sequence data. McrA protein was found to be localized in the outer membrane of Escherichia coli. To our knowledge this is the first restriction enzyme localized in the outer membrane of Escherichia coli.

EVALUATION OF THE FEASIBILITY OF USING LACTOSE AS THE INDUCER IN PRODUCING THE VIRAL ENHANCING FACTOR FROM HELICOVERPA ARMIGERA GRANULOSIS VIRUS IN ESCHERICHIA COLI BL21DE3 / 微生物学通报

Lactose was shown to no less competent than Isopropyl-?-D-thiogalactoside (IPTG) in inducing the expression of the ENHANCIN coding gene from Helicoverpa armigera granulosis virus in Eswcherichia coli BL21 (DE3) regulated by a T7 promoter, since the lactose induction could lead to an ENHANCIN band no smaller than the one in IPTG induction on the SDS-PAGE gel. This would decrease the cost of the large-scale ENHANCIN production. The lactose concentration was optimized at 2.2% - 2.5% (w/v) . Different treatments on the lactose sterilization showed that lactose steam- sterilized in 116. 5℃ for 15min could lead to the ENHANCIN production. The convenience and the relatively low cost in its" operation could further decrease the cost of the ENHANCIN production.

Construction of a T7 phage display cDNA library from lung of Microtus fortis / 中国血吸虫病防治杂志

Objective To construct a T7 phage display cDNA library from the lung of Microtus fortis for further screening the schistosomiasis-resistence-related genes of Microtus fortis. Methods mRNA was isolated from total RNA extracted from the lungs of Microtus fortis by TRIzol reagent, and was used to synthesize double strain cDNA by the reverse transcription. Then the double strain cDNA was given with EcoRⅠ and Hind Ⅲ adhering ends by ligation with the directional EcoRⅠ/Hind Ⅲ linkers and digestion with EcoRⅠ and Hind Ⅲ. The double strain cDNA fragments longer than 300 bp in length were fractionated by the Mini Column, and ligated into the T7 Select 10-3b vector with EcoRⅠ and Hind Ⅲ adhering ends. After packaging in vitro, the recombinant T7 Select 10-3b was transformed into BLT5403 to construct a T7 phage display cDNA library. Results The library constructed here contained 1.5?106 clones and the titer of the amplied library was 1.1?1012 pfu/ml. The PCR identification results of 100 clones picked at random showed that 91% clones were recombinant and 90% of recombinant clones contained cDNA fragments longer than 300 bp in length. Conclusion A T7 phage display cDNA library from the lung of Microtus fortis is successfully constructed.