Cloning of two plastid division ftsZ genes from Nicotiana tabacum and their expression in E.coli

Two cDNAs of plastid division gene NtFtsZ1-1 and NtFtsZ1-2 are isolated from Nicotiana tabacum by RT-PCR and rapid amplification cDNA ends (RACE) method. Analysis of the deduced amino acid sequences encoded by NtFtsZ1-1 and NtFtsZ1-2 indicate that these two proteins possess the typical conservative motifs and GTP binding sites existing in all FtsZ proteins. The existence of putative plastid transit peptide in their N-terminal suggests that there are at least two transit-peptide containing FtsZ proteins in higher plants. Phylogenetic analysis based on amino acid sequences of FtsZ proteins also supports this interference. These two NtFtsZ genes demonstrate a similar expression pattern during the plant development, detected by Northern blot. Expression of NtFtsZ1-1 and NtFtsZ1-2 in E.coli interrupts the normal division process of host cells. These results suggest the diverse functions of FtsZ proteins in higher plants.     

大肠杆菌二硫键异构酶DsbA和脯氨酸异构酶PPIaseA双顺反子表达载体的构建及其在大肠杆菌中的表达

用ＰＣＲ方法获得大肠杆菌二硫键异构酶ＤｓｂＡ的编码基因ｄｓｂＡ和大肠杆菌脯氨酸异构酶ＰＰＩａｓｅＡ的编码基因ｒｏｔ,并将ＤｓｂＡ和ｒｏｔ以双顺反子形式克隆至含有Ｐｔａｃ启动子的表达载体ｐＫＫ２３３－２中。在ＩＰＴＧ的诱导下,ＤｓｂＡ和ＰＰＩａｓｅＡ获得了表达。ＳＤＳ－ＰＡＧＥ和薄层扫描分析表明：ＤｓｂＡ、ＰＰＩａｓｅＡ的表达水平分别为占菌体裂解上清液总蛋白质的４．３２％和４．０６％。     

苜蓿银纹夜蛾核型多角体病毒Gp64蛋白的优化表达

研究培养基成份、pH值、接种量、诱导温度、诱导时间及诱导剂浓度等条件对苜蓿银纹夜蛾核型多角体病毒Gp64蛋白在大肠杆菌BL21(DE3)中表达水平的影响.SDS-PAGE电泳分析结果表明:在37℃的LB培养基中培养3h,用终浓度为0.3mmoL/L的IPTG于30℃诱导5h时表达水平最高.苜蓿银纹夜蛾核型多角体病毒Gp64重组蛋白在大肠杆菌中的优化表达为深度解析其在病毒感染过程中的功能奠定了基础.     

Nucleotide sequence of the hepatitis B virus genome (subtype ayw) cloned in E. coli.

The complete nucleotide sequence of hepatitis B virus genome (subtype ayw) cloned in Escherichia coli has been determined using the Maxam and Gilbert method and the dideoxynucleotide method. This sequence is 3,182 nucleotides long. Location of the nonsense codons shows that the coding capacity of the L chain is larger than the coding capacity of the S chain. Eight open regions, able to code for polypeptide chains larger than 100 amino acids, have been located. Region 6, which is the largest, covers more than 80% of the genome. The gene S which codes for polypeptide I of the Hbs Ag and was previously located between coordinates 95.1 and 73.6 is contained in region 7.     

枯草杆菌启动基因在大肠杆菌中的克隆和表达

以大肠杆菌启动基因选择载体pHE5为载体,枯草杆菌染色体DNA为供体,克隆了一批启动基因功能片段,带克隆片段的重组质粒在大肠杆菌中表现不同的四环素抗性水平,其中50%在100μg/ml以上,12%达到200μg/ml。对其中一个转化子进行质粒检测和分析,获得一个重组质粒pHE273,经酶切分析证明,克隆的强启动基因位于2.2kb的EcoRI-HindⅢ片段上。     

突变人GM-CSF基因在大肠杆菌中的表达及纯化

目的构建重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)的高效表达质粒,将其表达产物用于分离纯化的研究。方法用人外周血单核细胞获得总RNA作为模板和修饰的5′端密码子的引物经RT-PCR反应扩增到人GM-CSF基因,插入温控表达载体pDH构建了突变的hGM-CSF表达质粒,并将在E.coli中获得表达的突变rhGM-CSF包含体8 mol/L脲提取液进一步用离子交换色谱柱分离纯化。结果rhGM-CSF在大肠杆菌表达占菌体总蛋白量的22%,用弱阴离子交换色谱对rhGM-CSF的工程菌表达产物8 mol/L脲提取液直接经35 m in分离纯化,所得产物纯度可达95%,质量回收率可达到60%。结论这表明用离子交换色谱可进行rhGM-CSF的复性与同时纯化。     

Hepatitis B virus DNA integration in a sequence homologous to v-erb-A and steroid receptor genes in a hepatocellular carcinoma

Hepatitis B virus (HBV) is clearly involved in the aetiology of human hepatocellular carcinoma (HCC) and the finding of HBV DNA integration into human liver DNA in almost all HCCs studied suggested that these integrated viral sequences may be involved in liver oncogenesis. Several HBV integrations in different HCCs and HCC-derived cell lines have been analysed after molecular cloning without revealing any obvious role for HBV. From a comparison of a HBV integration site present in a particular HCC with the corresponding unoccupied site in the non-tumorous tissue of the same liver, we now report that HBV integration places the viral sequence next to a liver cell sequence which bears a striking resemblance to both an oncogene (v-erb-A) and the supposed DNA-binding domain of the human glucocorticoid receptor and human oestrogen receptor genes. We suggest that this gene, usually silent or transcribed at a very low level in normal hepatocytes, becomes inappropriately expressed as a consequence of HBV integration, thus contributing to the cell transformation.     

人胰岛素A链与B链基因的化学合成,克隆与表达

采用大肠杆菌密码子，用化学法分别合成人胰岛素２１个氨基酸的Ａ链，３０个氨基酸的Ｂ链，并在基因的５端引入甲硫氨酸密码子，尾部加入终止密码，两端加入限制性内切酶，Ａ链为８３个核苷酸，Ｂ链为１１０个核苷酸，经纯化后的寡聚核苷酸进行酶促连接反应后，分别克隆了人胰岛素Ａ、Ｂ链基因，然后选用ＰＬ启动子，构建了以牛生长前１３４个氨基酸的大片段基因，分别与人胰岛素Ａ链、Ｂ链基因的融合基因的融合基因的表达质粒ＰＬＷ     

Neurospora NADP-glutamate dehydrogenases and its expression in E. coli and transgenic plants

Genes of NADP-glutamate dehydrogenase (NADP-GDH) were cloned from Neurospora intermedia (Ni), N. crassa (Nc), and N. sitophila (Ns). The sequences showed a high degree of homology at the cDNA and protein level. The three GDH genes were cloned into pET30a and expressed in E. coli. The activity assay of purified GDH showed that the Ni-GDH had a higher activity and affinity to ammonia than Ns-GDH, and Nc-GDH. The K_m value of Ni-GDH ranges from 0.3 to 0.45 mmol/L. Ni-gdh gene was transformed to Nicotiana bethamiana plants. The transformed plants grew much better in low nitrogen media than the only ROKII vector transformed control.     

小鼠酪氨酸酶的克隆及在大肠杆菌中的表达

采用逆转录-聚合酶链式反应(RT -PCR)方法,从小鼠黑色素瘤总RNA中扩增得到小鼠酪氨酸酶的cDNA .该基因重组到T7启动子控制下的表达载体pET - 2 2b(+)中,构建表达质粒pET -TYR并转化大肠杆菌Rosetta (DE3) .SDS -PAGE及蛋白质序列测定表明,经0 .8mmol/L异丙基硫代- β-D -半乳糖苷(IPTG)诱导后,可高效表达重组的小鼠酪氨酸酶,表达量约占菌体总蛋白质的2 5 % ,表达产物为包涵体     

乙型肝炎病毒标志物与肝功能相关分析

目的探讨乙型肝炎病毒标志物与肝功能的关系.方法对比分析乙型肝炎102例病人中大三阳(HB-sAg、HBeAg、HBcAb阳性)44例、小三阳(HBsAg、HBeAb、HBcAb阳性)58例的谷丙转氨酶(ALT)、谷革转氨酶(AST)及总胆红素(T-BIL)浓度及白蛋白/球蛋白(A/C)异常情况.结果大三阳组ALT为(345.72±198.40)u/L,AST为(335.63±169.61)u/L,T-BIL为(78.68±26.78)μmol/L,A/G异常率为59.09%;小三阳组ALT为(155.63±98.53)u/LAST为(161.27±89.50)u/L,T-BIL为(38.68±16.78)μmol/L,A/G异常率为29.31%.两组比较有显著性差异(P＜0.05).结论乙型肝炎的病毒标志物与肝功能损害有关,大三阳病人的肝功损害比小三阳的明显严重.     

乙型肝炎病毒标志物与肝功能相关分析

目的探讨乙型肝炎病毒标志物与肝功能的关系.方法对比分析乙型肝炎102例病人中大三阳(HB-sAg、HBeAg、HBcAb阳性)44例、小三阳(HBsAg、HBeAb、HBcAb阳性)58例的谷丙转氨酶(ALT)、谷革转氨酶(AST)及总胆红素(T-BIL)浓度及白蛋白/球蛋白(A/C)异常情况.结果大三阳组ALT为(345.72±198.40)u/L,AST为(335.63±169.61)u/L,T-BIL为(78.68±26.78)μmol/L,A/G异常率为59.09%;小三阳组ALT为(155.63±98.53)u/LAST为(161.27±89.50)u/L,T-BIL为(38.68±16.78)μmol/L,A/G异常率为29.31%.两组比较有显著性差异(P＜0.05).结论乙型肝炎的病毒标志物与肝功能损害有关,大三阳病人的肝功损害比小三阳的明显严重.     

人IFN-γ在大肠杆菌中高效表达和色谱复性研究

从健康人外周血分离单核细胞，经LPS刺激后提取总RNA，用RT—PCR扩增hIFN-γ的cD—NA。将扩增产物克隆后进行序列分析证明，克隆的hIFN-γcDNA除存在2个序列多态性位点外，与天然的干扰素氨基酸序列完全一致。利用温控表达系统在大肠杆菌中实现了hIFN-γ的高效表达，表达的目的蛋白可占茵体总蛋白的55％。用5L和50L发酵罐放量生产的产物，经高效疏水色谱HPHIC直接复性和纯化得到了重组hIFN-γ纯品，其比活性与天然蛋白相近．