Probing conformational change of T7 RNA polymerase and DNA complex by solid-state nanopores

Proteins are crucial to most biological processes, such as enzymes, and in various catalytic processes a dynamic motion is required. The dynamics of protein are embodied as a conformational change, which is closely related to the flexibility of protein. Recently, nanopore sensors have become accepted as a low cost and high throughput method to study the features of proteins. In this article, we used a SiN nanopore device to study the flexibility of T7 RNA polymerase（RNAP） and its complex with DNA promoter. By calculating full-width at half-maximum（FWHM） of Gaussian fits to the blockade histograms, we found that T7 RNAP becomes more flexible after binding DNA promoter. Moreover, the distribution of fractional current blockade suggests that flexibility alters due to a breath-like change of the volume.

Promoting the production of challenging proteins via induced expression in CHO cells and modified cell-free lysates harboring T7 RNA polymerase and mutant eIF2α

Chinese hamster ovary(CHO)cells are crucial in biopharmaceutical production due to their scalability and capacity for human-like post-translational modifications.However,toxic proteins and membrane proteins are often difficult-to-express in living cells.Alternatively,cell-free protein synthesis can be employed.This study explores innovative strategies for enhancing the production of challenging proteins through the modification of CHO cells by investigating both,cell-based and cell-free approaches.A major result in our study involves the integration of a mutant eIF2 translation initiation factor and T7 RNA polymerase into CHO cell lysates for cell-free protein synthesis.This resulted in elevated yields,while eliminating the necessity for exogenous additions during cell-free production,thereby substantially enhancing efficiency.Additionally,we explore the potential of the Rosa26 genomic site for the integration of T7 RNA polymerase and cell-based tetracycline-controlled protein expression.These findings provide promising advancements in bioproduction technologies,offering flexibility to switch between cell-free and cell-based protein production as needed.

A New Tandem Gene Construction Method Involving a Cloning System Using <i>Poxvirus DNA polymerase</i>, and Its Application to Gene Expression

A simple method for constructing polymerized genes using only restriction enzymes and commercially available cloning systems was established. In this system, gel isolations or purifications of target genes after restriction enzyme digestions or PCR amplifications, which often cause errors and mutations in the target gene sequence, are not necessary. To verify the usefulness of this method, one, two, four, eight, and sixteen tandem-repeats of the Green Fluorescent Protein (GFP) expression gene in Escherichia coli were sequentially constructed. Efficacies of the GFP gene expression of those plasmids in E. coli showed an increasing trend in accordance with the copy numbers of the gene. On SDS polyacrylamide gel electrophoresis with Coomassie blue staining, no expressed protein could be seen in E. coli cells harboring plasmids that contained one or two copies of the gene. However, expressed protein bands in E. coli cells were clearly detected with 4 copies of the gene. In quantitative analyses involving green fluorescence intensities per culture volume, the expression level in E. coli with 16 copies of the gene was 36.3-fold higher than that in E. coli with one copy at 22 hours after induction.

Rapid Recovery of Classical Swine Fever Virus Directly from Cloned cDNA

The reverse genetics for classical swine fever virus （CSFV） is currently based on the transfection of in vitro transcribed RNA from a viral genomic cDNA clone, which is inefficient and time-consuming. This study was aimed to develop an improved method for rapid recovery of CSFV directly from cloned cDNA. Full-length genomic cDNA from the CSFV Shimen strain, which was flanked by a T7 promoter, the hepatitis delta virus ribozyme and T7 terminator sequences, was cloned into the low- copy vector pOK12, producing pOKShimen-RzTФ. Direct transfection of pOKShimen-RzTqb into PK/T7 cells, a PK-15- derived cell line stably expressing bacteriophage T7 RNA polymerase, allowed CSFV to be rescued rapidly and efficiently, i.e., at least 12 h faster and 31.6-fold greater viral titer when compared with the in vitro transcription-based rescue system. Furthermore, the progeny virus rescued from PK/T7 cells was indistinguishable, both in vitro and in vivo, from its parent virus and the virus rescued from classical reverse genetics. The reverse genetics based on intracellular transcription is efficient, convenient and cost-effective. The PK/T7 cell line can be used to rescue CSFV directly from cloned cDNA and it can also be used as an intracellular transcription and expression system for studying the structure and function of viral genes.

T7 RNA polymerase transcription of Escherichia coli isoacceptors tRNA^(Leu)

The T7 promoter and the gene encoding tRNAleu were synthesized chemically and cloned into plasmid pUC19, where the gene was transcribed in vitro with T7 RNA polymerase purified. It was found that spermidine had a negative effect on T7 transcription. The amount of transcribed tRNA was about 250 times that of the template DNA under optimal reaction conditions. The unmodified tRNAleu1 and tRNAleu2 had similar leucine accepting ability, which however was only one fourth that of the native modified ones. This result indicated that the modified nucleotides of tRNAleu played some though not key roles in its aminoacylation.

联合检测外周血及腹水中CK20水平对结直肠癌微转移的意义

目的检测患者术前外周血和腹水中细胞角蛋白(CK20)的水平对结直肠癌微转移的意义。方法采用改进的T7-RNA聚合酶催化的荧光扩增法(FACTT-T7 RNA),通过与酶联免疫法(ELISA)和流式细胞学技术(FCM)的对比,对附属第三医院2012年11月至2013年11月收治的56例结直肠癌患者术前外周血和术中腹水的CK20水平进行检测评价。结果 56例结直肠癌患者术中,外周血CK20阳性率FACTT为57.14%(32/56),ELISA为25.00%(14/56);腹水CK20阳性率FACTT为30.36%(17/56),FCM为14.29%(8/56),组内对比差异具有统计学意义(P<0.05);FACTT方法检测外周血联合腹水CK20阳性检出率达73.21%(41/56),组间对比率差异具有显著统计学意义(分别为P<0.05,P<0.01),CK20的表达与肿瘤TNM分期、分化程度有关(P<0.05)。结论 FACTT方法联合检测外周血和腹水中CK20水平的阳性表达率高,对结直肠癌患者的微转移具有重要的临床意义。

Establishment of a coupled expression system mediated by modified T7 RNA poly-merase gene

A coupled expression system for plants was established in this study. The 5’-terminal of T7 RNA poly-merase gene was modified by addition of the coding sequence of nuclear location signal from SV40 large T antigen. Plant expression vector pBBT7 was constructed with the modified T7 RNA polymerase gene under the control of CaMV35S promoter. Another expression vector pBTG contained cassette of gusA controlled by T7 promoter. The two vectors were co-transformed into tobacco via the Agrobecte-rium -mediated method. Results of GUS activity indicated that the co-transformed plant with pBBT7 and pBTG showed a high level of GUS activity. The results demonstrated that the coupled expression system of T7 polymerase and T7 promoter was workable in plants.

Inhibition of vascular endothelial growth factor gene expression by T7-siRNAs in cultured human retinal pigment epithelial cells

Background Retinal pigment epithelial (RPE) cells play an important role in the occurrence of choroidal neovascularization (CNV). Vascular endothelial growth factor (VEGF) as a positive regulatory growth factor is produced by the RPE in an autocrine or paracrine manner, promoting CNV development. Duplexes of 21 nt RNAs, known as short interfering RNAs (siRNAs), efficiently inhibit gene expression by RNA interference when introduced into mammalian cells. We searched for an efficient siRNA to interfere with VEGF expression in RPE cells and shed light on the treatment of CNV.Methods Human primary RPE (hRPE) cells were cultured and identified. Three pairs of siRNAs were designed according to the sequence of VEGF 1-5 extrons and synthesized by T7 RNA polymerase transcription in vitro. To evaluate the inhibitory activity of T7-siRNAs, hRPE cells were transfected via siPORT Amine. The interfering effect of T7-siRNAs in hRPE cells was examined by semiquantitative reverse transcription-polymerase chain reaction and immunofluorescence. Results Three pairs of T7-siRNAs synthesized by in vitro transcription with T7 RNA polymerase suppressed VEGF gene expression with efficiency from 65% to 90%. T7-siRNA (B), targeted region at 207 nt to 228 nt and double stranded for 21 nt with 2 nt UU 3’ overhangs, was the most effective sequence tested for inhibition of VEGF expression in hRPE cells. Compared with nontransfected cells, the mean fluorescence in hRPE cells transfected with T7-sRNAs was significantly less (P<0.01). siRNA with a single-base mismatch and ssRNA(+) did not show suppressing effect. Furthermore, it was found that siRNAs had a dose dependent inhibitory effect (5 to 10 pmol).Conclusion T7-siRNA can effectively and specifically suppress VEGF expression in hRPE cells and may be a new way to treat CNV.

支气管哮喘患者记忆CD_4^+T淋巴细胞活化相关基因的研究

目的筛选及鉴定支气管哮喘(简称哮喘)患者记忆 CD_4^+T 淋巴细胞活化相关基因。方法使用差异显示聚合酶链反应(DDPCR)方法筛选哮喘患者和健康人记忆 CD_4^+T 淋巴细胞差异表达基因,采用逆转录-聚合酶链反应(RT-PCR)法检测差异表达基因的 mRNA 表达水平。统计学处理采用 SPSS 10.0软件。计量资料采用±s表示,组间比较采用双因素方差分析,P<0.05为差异有统计学意义。结果经 DDPCR 克隆获得了19条差异片段,经同源性分析显示其中2条片段分别与白细胞介素-7(IL-7)和 MM-1基因高度同源,采用 RT-PCR 检测进一步证实了这2个基因在哮喘患者激活的记忆 CD_4^+T 淋巴细胞中表达白细胞介素-7和 MMI 基因激活后分别为0.390±0.029、0.629±0.047(F 值分别为983、1264,P 均<0.05)。结论哮喘患者激活的记忆 CD_4^+T 淋巴细胞中 IL-7和MM-1基因的上调表达可能是哮喘患者与健康人对于变应原出现不同反应的分子机制之一。

Establishment of a Reverse Genetic System of Severe Fever with Thrombocytopenia Syndrome Virus Based on a C4 Strain

Severe fever with thrombocytopenia syndrome virus(SFTSV)is an emerging tick-borne bunyavirus that causes hemorrhagic fever-like disease(SFTS)in humans with a case fatality rate up to 30%.To date,the molecular biology involved in SFTSV infection remains obscure.There are seven major genotypes of SFTSV(C1-C4 and J1-J3)and previously a reverse genetic system was established on a C3 strain of SFTSV.Here,we reported successfully establishment of a reverse genetics system based on a SFTSV C4 strain.First,we obtained the 5’-and 3’-terminal untranslated region(UTR)sequences of the Large(L),Medium(M)and Small(S)segments of a laboratory-adapted SFTSV C4 strain through rapid amplification of cDNA ends analysis,and developed functional T7 polymerase-based L-,M-and S-segment minigenome assays.Then,fulllength cDNA clones were constructed and infectious SFTSV were recovered from co-transfected cells.Viral infectivity,growth kinetics,and viral protein expression profile of the rescued virus were compared with the laboratory-adapted virus.Focus formation assay showed that the size and morphology of the foci formed by the rescued SFTSV were indistinguishable with the laboratory-adapted virus.However,one-step growth curve and nucleoprotein expression analyses revealed the rescued virus replicated less efficiently than the laboratory-adapted virus.Sequence analysis indicated that the difference may be due to the mutations in the laboratory-adapted strain which are more prone to cell culture.The results help us to understand the molecular biology of SFTSV,and provide a useful tool for developing vaccines and antivirals against SFTS.

Homogeneous label-free fluorescent assay of small molecule-protein interactions using protein binding-inhibited transcription nanomachine

Quantitative analysis of interactions between small molecules and proteins is a central challenge in chemical genetics, molecular diagnostics and drug developments. Here, we developed a RNA transcription nanomachine by assembling T7 RNA polymerase on a small molecule-labeled DNA heteroduplex. The nanomachine, of which the RNA transcription activity can be quantitatively inhibited by protein binding, showed a great potential for small molecule-protein interaction assay. This finding enabled us to develop a novel homogeneous label-free strategy for assays of interactions between small molecules and their protein receptors. Three small molecule compounds and their protein receptors have been used to demonstrate the developed strategy. The results revealed that the protein-small molecule interaction assay strategy shows dynamic responses in the concentration range from 0.5 to 64 nM with a detection limit of 0.2 nM. Due to its label-free, homogeneous, and fluorescence-based detection format, besides its desirable sensitivity this technique could be greatly robust, cost-efficient and readily automated, implying that the developed small molecule-protein interaction assay strategy might create a new methodology for developing intrinsically robust, sensitive and selective platforms for homogeneous protein detection.