酿酒酵母嗜杀质粒(dsRNA)的提取与分离纯化

对酿酒酵母嗜杀质粒的提取、电泳检测及纯化方法进行了研究。以改进的Fried和Fink方法在不预破细胞壁的情况下,巯基乙醇在碱性条件(pH9. 3)预处理后,直接以SDS-苯酚抽提完整细胞分离到了嗜杀质粒(dsRNA) 。在此基础上, 以电泳透析方法获得了纯化的M2和L双链RNA,琼脂糖凝胶电泳测定分子大小分别为1.5kb和4.0kb。 Abstract:The method of extraction and purification of the double-stranded RNA killer plasmids from Saccharomyces cerevisiae was studied.With the improved method of Fried and Fink we directly extracted the intact cells with SDS-phenol after pre-treating the cells with ß-mercaptoethanol under analkli condition.The result was detected and confirmed by the gel electrophoresis.On this basis we acquired the purified M2 and L-dsRNA by means of electrophoretic dialysis,their molecular lengths being 1.5kb and 4.0kb respectively.     

The Comparative and Functional Study between Two Construction Methods of shRNA Expression Vector Targeted LMP1 Gene Encoded by EBV

To look for a more stable and convenient way of constructing short hairpin RNA expression vectors targeting the latent membrane protein-1(LMP-1)encoded by Epstein-Barr virus(pshLMP1),and to study the inhibition function of pshLMP1 expression vectors in HNE1 cells,we designed the pshLMP1 expression cassette and pshLMP1 expression vectors by both the annealing method and PCR method and then co-transfected with pEGFP-N1-1158 into HNE1 cells to observe the mRNA and protein levels of LMP-1 genes by green fluorescence analysis,RT-PCR and western blot.pshLMP1 expression vectors were successfully obtained by both methods but better cloning efficiency was achieved and fewer deletions and mutations of nucleotides were achieved with the PCR method.Furthermore,the mRNA and protein levels of LMP-1 genes were down-regulated by pshLMP1 expression vectors.According to our research,we found that the PCR method provides a more efficient way to construct pshLMP1 expression vectors which have the ability to inhibit the function of LMP-1 genes expressed in HNE1 cells,and also provides a novel application of RNA interference technology against-EBV.     

Identification of differentially expressed genes of primary spermatocyte against round spermatid isolated from human testis using the laser capture microdissection technique

The method of laser capture microdissection (LCM) combined with suppressive subtractive hybridization (SSH) was developed to isolate specific germ cells from human testis sections and to identify the genes expressed during differentiation and development. In the present study, over 10,000 primary spermatocytes and round spermatid cells weresuccessfully isolated by LCM. Using the cDNAs from primary spermatocytes and round spermatids, SSH cDNAs library of primary spermatocyte-specific was constructed. The average insert size of the cDNA isolated from 75 randomly picked white clones was 500 bp, ranging from 250 bp to 1.7 kb. Using the dot-blot method, a total of 421 clones were examined, resulting in the identification of 390 positive clones emitting strong signals. Partial sequence of cDNAs prepared from each clone was determined with an overall success rate of 84.4%. Genes encoding cytochrome c oxidase Ⅱ and the rescue factor-humanin were most frequently expressed in primary spermatocytes, suggesting their roles involved in meiosis.     

Intracellular expression of a single-chain antibody directed against type IV collagenase inhibits the growth of lung cancer xenografts in nude mice

It was documented that type IV collagenase with two subtypes of 72 ku/MMP-2 and 92 ku/MMP-9 plays an important role in tumor invasion and metastasis. The endoplasmic reticulum (ER)- retained, single chain Fv antibody fragment (scFv) was used to inhibit the function of type IV collagenase. For expression in mammalian cells, the assembled scFv M97 gene with ER retention signal encoding 6 additional amino acids (SEKDEL) was reamplified by PCR. The amplified fragments were cloned into the pcDNA3.1 vector. The resulting plasmid was sequenced and then introduced into PG cells, a highly metastatic human lung cancer cell line, by lipofectAMINE method. The result of intrabody gene therapy showed that type IV collegenase expression was down regulated significantly as measured by ELISA. The biological behavior of PG cell, such as the ability of in vitro invasion through Matrigel, colony formation on soft agar, was also inhibited by scFv M97 transfection. Animal experiments in a xenograft model of human lung cancer     

Effect of LDL concentration polarization on the uptake of LDL by human endothelial cells and smooth muscle cells co-cultured

To substantiate our hypothesis that concentration polarization of low-density Upoprotein （LDL） plays an important role in the localization of atherogenesis, we investigated the effects of wall shear stress and water filtration rate （or perfusion pressure） on the luminal surface LDL concentration （cw） and the LDL uptake by human vascular endothelial cells and smooth muscle cells co-cultured on a permeable membrane using a parallel-plate flow chamber technique and a flow cyto-metry method. The results indicated that the uptake of fluorescent labeled LDL （DiI-LDL） by the co-cultured cells was positively correlated with Cw in a non-linear fashion. When cw was low, the uptake increased very sharply with increasing Cw. Then the increase became gradual and the uptake was seemingly leveled out when Cw reached beyond 160 μg/ml. The present study therefore has provided further experimental evidence that concentration polarization may occur in the arterial system and have a positive correlation with the uptake of LDLs by the arterial wall, which gives support to our hypothesis regarding the localization of atherogenesis.     

Inhibition of genes expression of SARS coronavirus by synthetic small interfering RNAs

RNA interference (RNAi) is triggered by the presence of a double-stranded RNA (dsRNA), and results in the silencing of homologous gene expression through the specific degradation of an mRNA containing the same sequence, dsRNAmediated RNAi can be used in a wide variety of eucaryotes to induce the sequence-specific inhibition of gene expression.Synthetic 21-23 nucleotide (nt) small interfering RNA (siRNA) with 2 nt 3‘ overhangs was recently found to mediate efficient sequence-specific mRNA degradation in mammalian cells. Here, we studied the effects of synthetic siRNA duplexes targeted to SARS coronavirus structural proteins E, M, and N in a cell culture system. Among total 26 siRNAduplexes, we obtained 3 siRNA duplexes which could sequence-specifically reduce target genes expression over 80% at the concentration of 60 nM in Vero E6 cells. The downregulation effect was in correlation with the concentrations of the siRNA duplexes in a range of 0-450 nM. Our results also showed that many inactive siRNA duplexes may be brought to life simply by unpairing the 5‘ end of the antisense strands. Results suggest that siRNA is capable of inhibiting SARS coronavirus genes expression and thus may be a new therapeutic strategy for treatment of SARS.     

酵母菌SH2发酵产物增强干扰素抗病毒活性 及其有效成分的初步研究

The fermentation broth of yeast strain SH-2 (FSH-2) which could enhance the biological potency of human interferon alpha (huIFN-α) was detected. Its enhancing ratio was 1.64～6.86 fold. A group of proteins was seperated and purified from the fermentation supernatant by chromatography on Sephadex G-75 and HPLC. Each protein showed the similar band on PAGE and SDS-PAGE. Their molecular weights ranged from 52 to 72 kD. The proteins were stainded with periodic acid Schiff's agent. Lowry's method and sulfuric acid-phenol method were respectively used to determine the contents of proteins and neutral sugars. The results showed the ratio of protein to sugar was 3∶1. These results indicated that the proteins were extracellular glycoproteins (YEGPs) secreted by yeast strain SH-2. YEGPs could significantly enhance the biological potency of huIFN-α. It was veritied by the experiment of cytopathic effect inhibition in Wish cells. The enhancing ratios were 1.6～2.8 fold and 1.4～4.0 fold, respectively. The enhancing ratio of the elution protein of FSH-2 seperated by Sephadex G-75 was 2.01～5.68 fold. YEGPs alone could not enhance the potency of huIFN-α and had no biological activity as IFNs.