欧芹苯丙氨酸脱氨酶cDNA在乳酸乳球菌中的表达研究

将欧芹（Ｐｅｔｒｏｓｅｌｉｎｕｍｃｒｉｓｐｕｍ）苯丙氨酸脱氨酶（ＰＡＬ）ｃＤＮＡ亚克隆到组成型表达载体ｐＭＧ３６ｅ启动子Ｐ３２下游,电穿孔法转化乳酸乳球菌,获得有ＰＡＬ表达活性的乳酸乳球菌工程菌（ｐＭＧ３６ｅＰＡＬ／Ｌ．ｌａｃｔｉｓＭＧ１３６３）。通过递归ＰＣＲ合成了一段１２０ｂｐ的调控片段,用以将ｐＭＧ３６ｅ改造为分泌型表达载体ｐＸＨＳ,以翻译偶联的方式表达ＰＡＬ,可使ＰＡＬ的Ｎ末端带上ｕｓｐ４５信号肽,结果亦检测到ＰＡＬ酶活性。自行分离克隆了乳酸乳球菌热休克蛋白基因ｄｎａＪ的启动子区域,构建了热诱导表达载体ｐＸＨＪ,获得ＰＡＬ热诱导表达工程菌（ｐＸＨＪＰＡＬ／Ｌ．ｌａｃｔｉｓＩＬ１４０３）,经３０℃至３７℃热诱导,可使ＰＡＬ表达活性提高至２倍。本文还就乳酸乳球菌ＰＡＬ工程菌在经典型苯丙酮尿症防治中的应用进行了分析和讨论     

人铜锌超氧化物歧化酶cDNA的克隆,测序及表达

用逆转录聚合酶链反应（ＲＴＰＣＲ）,以人胎肝组织总ＲＮＡ为模板,扩增了人铜锌超氧化物歧化酶（ｈＣｕ,ＺｎＳＯＤ）的ｃＤＮＡ,并进行序列分析,将该ｈＣｕ,ＺｎＳＯＤｃＤＮＡ重组到Ｔ７启动子控制下的分泌型表达载体ｐＥＴ２２ｂ（＋）中,构建表达质粒ｐＥＴＳＯＤ,并转化大肠杆菌ＢＬ２１（ＤＥ３）。ＳＤＳＰＡＧＥ及蛋白质印迹分析表明,经１ｍｍｏｌ／Ｌ异丙基硫代βＤ半乳糖苷（ＩＰＴＧ）诱导后,可高效表达一分子量为１９ｋＤ的蛋白质,与抗人ＳＯＤ多抗有特异的免疫反应,表达量约为菌体总蛋白质的３０％,具有特异性ＳＯＤ酶活性,酶活力可达１７９７ｕ／ｍｌ培基。     

粒细胞—巨噬细胞集落刺激因子／白细胞介素—3融合蛋白的表达研究

利用ＰＣＲ扩增得到粒细胞－巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）、白细胞介素－３（ＩＬ－３）完整基因片段,将其分别克隆ｐＧＥＭ－Ｔ构建成ＧＭ－ＣＳＦ／ＩＬ－３融合蛋白基因,ＤＮＡ序列与设计预期一致。将得到的融合蛋白基因克隆对７２ＲＮＡ聚合酶表达载体ｐＴ７ｚｚ,得到表达质粒ｐＦｕ,经转化至表达宿主Ｅ．ｃｏｌｉ ＢＬ２１（ＤＥ３）,在ＩＰＴＧ诱导下获得融合蛋白目的产物的直接表达。经ＳＤＳ－ＰＡＧＥ电泳鉴     

猪生长激素基因在杆状病毒载体系统中的表达

通过对猪生长激素（ｐＧＨ）基因的ｃＤＮＡ进行测序,得到ｐＧＨｃＤＮＡ的全序列,并与Ｓｅｅｂｕｒｇ等报道的序列进行了比较和讨论。然后利用具人工合成启动子和多角体蛋白ＸＩＶ启动子的转移载体质粒ｐＳＸＩＶＶＩ＾＋Ｘ３／４构建出含ｐＧＨ基因的重组质粒ｐＸ３／４－ｐＧＨ。将ｐＸ３／４－ｐＧＨ与致死缺失型线性化ＡｃＭＮＰＶ－ＯＣＣ＾－ＤＮＡ共转染Ｓｆ９细胞,构建出既能形成多角体又能表达ｐＧＨ基因的苜蓿丫纹夜蛾     

链霉菌—大肠菌穿梭质袜载体pSGLgpp的构建及应用

质粒ｐＳＧＬ１（７．４ｋｂ）是从球孢链霉菌（Ｓｔｒｅｐｔｏｍｙｃｅｓ ｇｅｏｂｉｓｐｏｒｕｓ）中分离得到的一个高拷贝质粒,已测定其最小复制子序列。从球孢链霉菌总ＤＮＡ中采用ＰＣＲ方法扩增获得编码Ｃ１０２７前蛋白信号肽的ＤＮＡ片断ｇｐｐ。将ｇｐｐ克隆至ｐＳＧＬ１的衍生质粒ｐＳＧＬＮ中,获得新的链霉菌表达型质粒载体ｐＳＧＬｇｐｐ。应用该质粒进行了人的河溶性白细胞介素１受体Ｉ型的表达。     

Egr-1基因调控序列连接OSM cDNA表达质粒的构建及其在黑色素瘤细胞中的表达

ＯＳＭ是一种对黑色素瘤细胞显示抑制作用的细胞因子．为进行ＯＳＭ针对黑色素瘤的基因－放射治疗研究,构建了小鼠Ｅｇｒ－１基因调控序列引导入ＯＳＭｃＤＮＡ真核表达质粒（ｐＥＯ）,ｐＥＯ质粒转染小鼠Ｂ－１６黑色素瘤细胞,经Ｇ４１８和抗人ＯＳＭ抗体的筛选,获得了稳定表达ＯＳＭ的克隆细胞（ｐＥＯ－１细胞）,ＯＳＭ表达量可达５．９７ｎｇ每１０５细胞天,分子量为３２ｋＤ．ｐＥＯ－１细胞用一定浓度Ｈ２Ｏ２处理后ＯＳＭ表达量可提高６２％,表明ｐＥＯ重组质粒可在氧自由基的刺激作用下增强ＯＳＭ表达     

小鼠金属硫蛋白 Ⅰ cDNA编码序列的克隆及其在昆虫细胞中的表达

将质粒ｐＢＸ－ＭＴ上的小鼠ＭＴ－ⅠｃＤＮＡ片段切下作为模板,通过ＰＣＲ方法删除该片段的非编码序列,将编码序列克隆到质粒ｐＢＳ－ＳＫ中,经ＤＮＡ序列测定后证明其克隆序列正确．再将ＭＴ－ⅠｃＤＮＡ编码序列插入到转移载体ｐＢａｃＰＡＫ８的ＢａｍＨⅠ和ＥｃｏＲⅠ位点之间,通过磷酸钙／ＤＮＡ共转染方法将其导入昆虫细胞Ｓｆ９中,以Ｗｅｓｔｅｒｎｂｌｏｔ和ＤｏｔＥＩＡ方法对表达产物进行了检测,表达量为１ｍｇ／Ｌ     

日本血吸虫26kD抗原基因在BCG中的表达

研究了外源基因日本血吸虫２６ｋＤ抗原（Ｓｊ２６ＧＳＴ）在卡介苗（ｂａｃｉｌｕｓＣａｌｍｅｔｅ－Ｇｕｅｒｉｎ,ＢＣＧ）、耻垢分枝杆菌（Ｍ．ｓｍｅｇｍａｔｉｓ）和大肠杆菌（Ｅ．ｃｏｌｉ）中的表达．运用重组ＤＮＡ和聚合酶链反应（ＰＣＲ）等分子生物学技术,以表达Ｓｊ２６ＧＳＴ的Ｅ．ｃｏｌｉｐＧＥＸ衍生质粒为模板,经ＰＣＲ得到编码Ｓｊ２６ＧＳＴ的全长ｃＤＮＡ片段．将其按正确的阅读框顺序,克隆到人结核杆菌热休克蛋白（ｈｅａｔｓｈｏｃｋｐｒｏｔｅｉｎ,ＨＳＰ）７０的启动子下游,再将ＨＳＰ７０启动子和Ｓｊ２６ＧＳＴ基因一起亚克隆到Ｅ．ｃｏｌｉ－分枝杆菌穿梭质粒ｐＢＣＧ－２０００中,得到Ｅ．ｃｏｌｉ－分枝杆菌穿梭表达质粒ｐＢＣＧ－Ｓｊ２６．ｐＢＣＧ－Ｓｊ２６电转化入ＢＣＧ和Ｍ．ｓｍｅｇｍａｔｉｓｍｃ２１５５中表达Ｓｊ２６ＧＳＴ抗原,所表达的天然重组Ｓｊ２６ＧＳＴ（ｒＳｊ２６ＧＳＴ）为可溶性蛋白,在ＳＤＳ－ＰＡＧＥ上分子量为２６ｋＤ处可见明显的表达蛋白带．其表达量分别占ＢＣＧ和Ｍ．ｓｍｅｇｍａｔｉｓ菌体总蛋白的１５％和１０％．可见,Ｓｊ２６ＧＳＴ基因能在ＢＣＧ中高效表达．     

抑瘤素M cDNA的克隆及表达研究

抑瘤素Ｍ是一种我功能的细胞生长调节因子,从ＰＭＡ刺激后的Ｕ９３７细胞系提取总ＲＮＡ,采用逆转录－ＰＣＲ方法分离到了抑瘤素Ｍ的ＣＤＮＡ;将抑瘤素Ｍ的ＣＤＮＡ克隆到质粒ＰＵＣ１９中,筛选三个阳性克隆进行序列分析,与国外报导序列完全一致;将抑瘤素Ｍ的ＣＤＮＡ克隆到质粒ＰＢＶ２２０后再转化ＤＨ５α进行模拟表达,ＳＤＳ－ＰＡＧＥ分析表明有ＯＳＭ表达,表达量约占细菌总蛋白５％,经过初步纯化的ＯＳＭ能明显抑制Ａ     

人血小板生成素受体c—MPL膜内部分的聚合作用

血小板生成素（ＴＰＯ）是调节血小板生成最主要的细胞因子,其生物学效应由其受体ｃ－ＭＰＬ介导。利用酵母双杂合系统研究ｃ－ＭＰＬ膜内部分在ＴＰＯ信号转导途径中的功能。首先用反转录ＰＣＲ（ＲＴ－ＰＣＲ）方法从人红白血病ＨＥＬ细胞系总ＲＮＡ中扩增并克隆Ｐ型ｃ－ＭＰＬ（ＭＰＬＰ）膜内部分ｃＤＮＡ,经测序验证后克隆至双杂合载体ｐＡＳ２和ｐＧＡＤ４２４中,重组质粒命名为ｐＡＳＭＭ和ｐＧＡＤＭＭ。将ｐＡＳＭＭ与ｐ     

人铜锌超氧化物歧化酶基因在乳酸乳球菌中的食品级表达

以lacF基因为食品级选择标记,构建了乳酸乳球菌食品级基因表达系统,并进而实现了人铜锌超氧化物歧化酶基因在乳酸乳球菌中的食品级表达。首先构建了含有lacF基因两侧同源DNA序列(0.5kb)的整合型质粒pUCEmDE,通过pUCEmDE与乳酸乳球菌MG5267染色体上单拷贝的乳糖操纵子之间的同源双交换,构建了lacF基因缺失突变的食品级受体菌WZ103 (Lac-),并经PCR及Lac表型检测所验证。然后构建了互补质粒pMG36eF,其lacF基因的表达受组成型的强启动子P32的控制。将其电转化导入WZ103后,Lac+表型得到恢复,表明WZ103中lacF基因的功能可被互补质粒pMG36eF上的lacF基因互补。随后,以互补质粒pMG36eF为基础,构建了不含任何抗生素抗性选择标记的人铜锌超氧化物歧化酶基因的食品级表达质粒pWZ104。通过非变性聚丙烯酰胺凝胶电泳和SOD活性凝胶染色分析,检测到WZ103(pWZ104)中Cu/Zn SOD的表达,并且具有生物活性。     

High-level expression of human extracellular superoxide dismutase in Escherichia coli and insect cells.

Much is known about the physical properties of the Cu,Zn- and Mn-superoxide dismutases (SODs). However, the biochemical characteristics and pharmacological properties of extracellular (EC)-SOD have been severely limited due to difficulties in obtaining and purifying the enzyme. The EC-SOD cDNA was inserted into the Escherichia coli expression plasmid pET-28a(+) which contains the T7 promoter and transformed into the E. coli BL21(DE3). After induction with 1 mmol/L isopropyl beta-D-thiogalactoside, the recombinant human EC-SOD was highly expressed as inclusion bodies. SDS-PAGE analysis revealed that recombinant EC-SOD accumulated up to 26% of the total soluble protein of E. coli cells. The expression product was purified by a Ni(2+)-IDA-Sepharose 6B column. After the denaturing and refolding processes, the recombinant human EC-SOD retains the specific enzymatic activity of 920 U/mg of the purified product. The gene encoding human EC-SOD mature peptide was also inserted into the donor plasmid pFastBacHTb. After transposition, transfection, and amplification were performed, the recombinant baculoviruses infected the Tn-5B1-4 cells and EC-SOD was highly expressed in Tn-5B1-4 cells. SDS-PAGE and Western blot analysis revealed that the subunit molecular weight of the expression product is 28 kDa. Furthermore, recombinant human EC-SOD retains the enzymatic specific activity of 200 U/mg of the Tn-5B1-4 cell lysates.     

T7 启动子作用下人尿激酶原 cDNA 在大肠杆菌中的高效表达及分离纯化

化学合成的人尿激酶原ｃＤＮＡ克隆在表达质粒ｐＥＴ－１１ｄ中,在Ｔ７启动子的作用下,经０．１ｍｍｏｌ／Ｌ ＩＰＴＧ诱导,在大肠杆菌ＢＬ２１（ＤＥ３）ｐＬｙｓＳ中获得表达。其表达量占菌体总蛋白的１５％,表达产物以无活性的包涵体形式存在。经体外变复性,Ｚｎ＾２＋选择性沉淀,抗体亲和柱层析,及Ｂｅｎｚａｍｉｄｉｎｅ亲和吸附,所表达的人尿激酶原被纯化为单一条带,其比活约１１００００ＩＵ／ｍｇ。     

Somatic homologous recombination in planta: The recombination frequency is dependent on the allelic state of recombining sequences and may be influenced by genomic position effects

The Escherichia coli sodA gene encoding the antioxidant enzyme Mn-containing superoxide dismutase (MnSOD), was cloned in the expression vector pMG36e. This vector has a multiple cloning site down-stream of a promoter and Shine-Dalgarno sequences derived from Lactococcus. The protein-coding region of sodA from E. coli was amplified by the polymerase chain reaction, using a thermocycler and Taq DNA polymerase before cloning into pMG36e. When introduced into E. coli, the recombinant plasmid expressed the predicted fusion protein, both in the presence and absence of oxygen. The expression of the fusion protein in E. coli was verified by SOD assays, activity gels and Western blots. The recombinant plasmid was also introduced into Lactococcus lactis, which contains a resident SOD, and into Lactobacillus gasseri, which is devoid of SOD. Transformed lactococci expressed an active SodA fusion protein plus an active hybrid protein composed of subunits of the Lactococcus and the recombinant E. coli enzymes. Transformants of L. gasseri expressed only the fusion SodA protein, which was enzymatically active.     

Somatic homologous recombination in planta: The recombination frequency is dependent on the allelic state of recombining sequences and may be influenced by genomic position effects

The Escherichia coli sodA gene encoding the antioxidant enzyme Mn-containing superoxide dismutase (MnSOD), was cloned in the expression vector pMG36e. This vector has a multiple cloning site down-stream of a promoter and Shine-Dalgarno sequences derived from Lactococcus. The protein-coding region of sodA from E. coli was amplified by the polymerase chain reaction, using a thermocycler and Taq DNA polymerase before cloning into pMG36e. When introduced into E. coli, the recombinant plasmid expressed the predicted fusion protein, both in the presence and absence of oxygen. The expression of the fusion protein in E. coli was verified by SOD assays, activity gels and Western blots. The recombinant plasmid was also introduced into Lactococcus lactis, which contains a resident SOD, and into Lactobacillus gasseri, which is devoid of SOD. Transformed lactococci expressed an active SodA fusion protein plus an active hybrid protein composed of subunits of the Lactococcus and the recombinant E. coli enzymes. Transformants of L. gasseri expressed only the fusion SodA protein, which was enzymatically active.     

Expression, purification, and characterization of His-tagged penicillin G acylase from Kluyvera citrophila in Escherichia coli

The DNA fragment encoding Kluyvera citrophila penicillin G acylase (KcPGA) was amplified and cloned into the vector pET28b to obtain a C-terminus His-tagged fusion expression plasmid. The fusion protein KcPGA was successfully overexpressed in Escherichia coli BL21(DE3). The optimal induction concentration of isopropylthio-beta-D-galactoside (IPTG) was found to be 5 microM. The fusion protein was purified in a single step by Ni-IDA affinity chromatograph to a specific activity of 35.3U/mg protein with a final yield of 89% representing a 23-fold purification. The data presented here suggest that the purified fusion protein is stable with respect to pH and temperature. The optimal pH and temperature of recombinant KcPGA are 8.5 and 55 degrees C, respectively. The Km and Vmax are 17.6 microM and 23.8 U/mg, respectively. Therefore, the high yield and high specific activity of recombinant KcPGA produced in E. coli, together with other kinetic parameters, represent an excellent basis for further development of recombinant KcPGA as an immobilized biocatalyst for industrial applications.     

南极假丝酵母脂肪酶B(CALB)基因在乳酸乳球菌MG1363中的整合及表达

根据南极假丝酵母脂肪酶B (CALB)的基因序列将CALB基因进行TA克隆、酶切鉴定及测序后,亚克隆至大肠杆菌-乳酸乳球菌穿梭表达栽体pMG36e-Nisl中,构建重组表达栽体pMG36e-Nisl-CALB.设计特异性引物P3和P4,对重组质粒pMG36e-NisI-CALB进行红霉素抗性基因的敲除,以构建食品级表达载体pMG36N-CALB,后再将两种重组质粒分别电转化入乳酸乳球菌MG1363,以Nisin为选择压力,考察CALB在MG1363中的表达情况.结果显示,成功构建了表达载体pMG36e-NisI-CALB及pMG36N-CALB,两株重组菌在含有20 IU Nisir/mL的培养基中均生长情况良好,遗传性能稳定,且经水解圈鉴定,CALB能够进行活性表达.进一步研究发现,CALB基因整合到乳酸乳球菌MG1363染色体中.     

Characterization of two copper/zinc superoxide dismutases (Cu/Zn-SODs) from the desert beetle Microdera punctipennis and their activities in protecting E. coli cells against cold

Superoxide dismutases (SODs) are crucial in scavenging reactive oxygen species (ROS); however, studies regarding SOD functions in insects under cold conditions are rare. In this paper, two novel Cu/Zn-SOD genes in the desert beetle Microdera punctipennis, an extracellular copper/zinc SOD (MpecCu/Zn-SOD) and an intracellular copper/zinc SOD (MpicCu/Zn-SOD), were identified and characterized. The results of quantitative real-time PCR showed that MpecCu/Zn-SOD expression was significantly up-regulated by 4 °C exposure for 0.5 h, but MpicCu/Zn-SOD was not. Superoxide anion radical (O₂•^-) content in beetles under 4 °C exposure for 0.5 h showed an initial sharp increase and fluctuated during the cold treatment period, which was consistent with the relative mRNA level of MpecCu/Zn-SOD. The total SOD activity in the beetle was negatively correlated to the O₂•^- content with a correlation coefficient of −0.437. An E. coli system was employed to study the function of each MpCu/Zn-SOD gene. The fusion proteins Trx-His-MpCu/Zn-SODs were over expressed in E. coli BL21 using pET32a vector, and identified by SDS-PAGE and Western blotting. The transformed bacteria BL21(pET32a-MpecCu/Zn-SOD) and BL21(pET32a-MpicCu/Zn-SOD) showed increased cold tolerance to −4 °C as well as increased SOD activity compared to the control BL21(pET32a). The relative conductivity and malondialdehyde content in the two MpCu/Zn-SODs transformed bacteria under −4 °C were significantly lower than the control BL21(pET32a). Furthermore, BL21(pET32a-MpecCu/Zn-SOD) had significantly higher SOD activity and cold tolerance than BL21(pET32a-MpicCu/Zn-SOD) under −4 °C treatment, and had lower conductivity than BL21(pET32a-MpicCu/Zn-SOD). In conclusion, low temperature led to the accumulation of O₂•^- in M. punctipennis, which stimulated the expression of extracellular MpCu/Zn-SOD gene and the increase of total SOD activity. E. coli overexpressing Trx-His-MpCu/Zn-SODs increased resistance to cold treatment-induced oxidative stress. Our findings will be helpful in further study of Cu/Zn-SOD genes in insect cold-tolerance.