A链链内二硫键移位突变胰岛素原的构建与表达

采用定点突变技术,将人胰岛素原基因编码框中A11位氨基酸残基与A12位氨基酸残基进行了互换,构建了[CysA11Ser,SerA12Cys]人胰岛素原突变基因.在大肠杆菌原核表达系统中对突变体基因进行了表达,并对表达产物进行了变复性研究及初步的分离纯化,获得了具有稳定结构的突变体蛋白质,探讨了突变体A链链内二硫键的形成状况,为进一步深入揭示A链链内二硫键的角色与功能打下了基础.     

关于(A6,A11)-双位点突变胰岛素原作为外源肽呈递骨架可行性的研究

通过重组DNA技术,以(A6,A11)-双突变胰岛素原分子为支架构建了含有外源抗栓肽功能区的嵌合分子,并在大肠杆菌进行了表达和纯化.对嵌合分子进行胰岛素受体结合活性、放射免疫活性及抗血小板聚集活性分析,结果表明该嵌合分子具有较强的血小板聚集抑制活性,而无胰岛素生物活性,证明以(A6,A11)-双突变胰岛素原作为外源肽序列呈递骨架是可行的.     

抗栓胰岛素原钙调素重组融合蛋白表达条件的摸索

以无活性人胰岛素原突变体为分子支架,在C肽位置嵌合特异拮抗血小板GPⅡbⅢa受体的去整合素活性结构位点序列,构建嵌合人胰岛素原突变体.该突变体相对分子质量大小为7.0×103,与钙调素的C末端融合后形成新型重组融合蛋白质,相对分子质量为24.7×103.构建该重组融合蛋白质的大肠杆菌BL21(DE3)表达系统,尝试在大肠杆菌表达体系中,对该重组融合蛋白质进行高效表达.结果表明,该重组融合蛋白质的表达率约占菌体全蛋白的30%,优化后的表达条件为25℃,50μmol.L-1诱导表达16 h.     

用点突变方法研究海参精氨酸激酶的活性位点

为研究动物能量代谢,用点突变的方法从双亚基海参(Stichopusjaponicus)精氨酸激酶中得到了3个突变体:C274A、R283G和H287A,并通过大肠杆菌E.coli表达。大部分精氨酸激酶都以包涵体的形式存在。经过纯化之后对3种突变体的催化活性和一些结构特征进行了分析。这3种突变体丧失了绝大部分催化活力,特别是突变体C274A,与野生型相比除了活性完全丧失之外,结构却几乎没有变化。这3个残基对海参精氨酸激酶结构或功能的保持有着重要作用,Cys274残基可能是海参精氨酸激酶的活性位点。     

Long R^3-IGF-Ⅰ的克隆表达

在重组质粒pExSecI-IGF-Ⅰ的基础上,采用基因克隆方法合成了长链人胰岛素样生长因子(Long R3-IGF-Ⅰ)基因序列,构建基于该基因的重组原核表达质粒.将重组质粒转入E.coli,并对其表达条件进行了优化.实验结果表明,在37 ℃,IPTG终浓度0.6 mmol/L,诱导3 h条件下,Long R3-IGF-Ⅰ以包涵体形式可得到高效表达.将包涵体变性溶解后通过分离纯化,目的蛋白纯度达到95%以上.通过尿素梯度透析复性法对变性包涵体复性,复性率达到66%.     

人尿激酶原纤原受体识别区嵌合突变体的构建与表达

采用同源转座方法,构建了在尿激酶原K区138～139位点插入了赖氨酸甘氨酸天冬氨酸(KGD)序列的2种尿激酶原嵌合体基因(proUK-KGDW, proUK-KGDS),并以昆虫细胞SF9为宿主获得了高效表达,表达量在33.34 μkat·mL-1, 细胞密度为106·mL-1,表达高峰期表达产物占细胞总蛋白的15%以上.表达产物展示出较高的纤溶活性,并具有良好的人尿激酶原免疫原性,同时,经SDS-PAGE试验检测,昆虫细胞分泌的突变体相对分子质量符合理论预期,为5.4×104,绝大多数为单链态形式.经过离子交换、分子筛层析及超滤浓缩等步骤对突变体蛋白进行了初步纯化,光浊度法试验表明含有KGDW序列的尿激酶原突变体具有较强的血小板聚集抑制活性,而含有KGDS序列的突变体几乎不具有任何活性,表明羧基端边侧序列对KGD展示正常的受体结合活性影响较大.溶解圈试验表明2种突变体均保留了较高的纤溶活性,说明插入序列不影响尿激酶原的酶切活性.     

胡萝卜抗冻蛋白在大肠杆菌中的高效表达

以pET—11a为载体构建了胡萝卜抗冻蛋白非融合表达的重组质粒，并在大肠杆菌菌株BL21(DE3，pLysS)中成功地进行了高效表达。表达的蛋白质以包涵体的形式存在，相对分子质量为36800。经正交试验得到了优化的诱导表达条件。包涵体经各种缓冲液洗涤纯化、溶解、复性和超滤浓缩后，经SDS—PAGE电泳检测，纯度达单一带水平。生物学活性研究表明，重组表达的胡萝卜抗冻蛋白具有很强的热滞活性和提高细菌耐寒性的作用。     

重组羧肽酶B在胰岛素原C肽制备工艺中的应用

用RT-PCR方法克隆了大鼠羧肽酶原B的cDNA编码序列,将其重组到原核表达质粒pT7-473中,并在大肠杆菌中以包涵体方式获得高表达。SDS-PAGE电泳及灰度扫描显示表达量达菌体总蛋白的35％,表达产物融合了表达质粒上的6His。在变性条件下,经Ni-NTA柱纯化,得到羧肽酶原B,在复性液中进行稀释复性后,胰蛋白酶切得具酶活性的羧肽酶B,然后经DEAE-FF分离纯化获得较纯的羧肽酶B(28．5mg／L),比活为13．5u／mg。用RP-HPLC分析胰蛋白酶 羧肽酶B酶解胰岛素原C肽三聚体的酶解产物,证明该重组的羧肽酶B可替代提取的羧肽酶B应用于胰岛素原C肽的制备工艺中。     

AiiA融合蛋白包涵体变性和复性的研究

通过IPTG诱导含有pGEXaiiA-B15质粒的工程菌使其大量表达AiiA融合蛋白.由于所表达的融合蛋白多为不可溶的包涵体,须经分离、变性溶解,再经过一个合适的复性过程才能实现变性蛋白的正确折叠,得到具有生物活性的蛋白.通过含N-十二烷基肌氨酸钠的缓冲液A溶液及尿素等变性剂使包涵体溶解,磷酸盐缓冲液透析复性.SDS-PAGE电泳表明包涵体已由不可溶转化成可溶的蛋白.抑菌试验证明复性后的AiiA蛋白对胡萝卜欧文氏软腐病菌引起的马铃薯软腐病有较明显的抑制作用.     

人胰岛素原类似物（B-Arg-Arg-A）基因的构建和表达

用聚合酶链反应（PCR）法将C肽为6个氨基酸残基的胰岛素原类似物基因删除突变成C肽仅为2个精氨酸残基的胰岛素原类似物（BArg-Arg-A）基因,即把pUC18BC'A改建为pUC18BR₂A。再将pUC18BR₂A与pJG105重组为表达质粒pJG107,使B-Arg-Arg-A与pJG105编码的一种多肽构成融合蛋白在大肠杆菌体系中表达。融合蛋白占细菌蛋白总量的58%。表达产物具有人胰岛素放射免疫活性。     

人胰岛素原密码子的优化及其在毕赤酵母中的表达

依据毕赤酵母偏好密码子,设计合成长效人胰岛素原( human proinsulin,HPI)基因序列,所合成的HPI基因全长为200 bp,并在其N端添加信号肽(EEAEAEAEPK)以提高目的蛋白表达.将改造后基因克隆到pPIC9K载体中,构建分泌表达型载体pPIC9K-HPI,转化到毕赤酵母(Pichia pastoris)GS115中.用遗传霉素G418梯度筛选获得高拷贝菌株,在甲醇诱导下表达分子质量为7.87 ku的HPI,利用金属螯合层析纯化蛋白质,胰蛋白酶酶切后利用人胰岛素试剂盒测得生物活性,HPI摇瓶最高产量可达35.4mg/L.该研究为获得大量长效胰岛素基因工程产品奠定研究基础.     

米曲霉中性蛋白酶高产菌株的复合诱变选育

利用紫外线和化学诱变剂硫酸二乙酯对米曲霉H10进行复合诱变,选育出产中性蛋白酶高产菌株H12-5。该菌株比对照菌株中性蛋白酶活力提高108.5%。     

弱化H+-ATPase的德氏乳杆菌乳酸亚种的生理特性研究

以德氏乳杆菌乳酸亚种（L5）为出发菌株,以新霉素为选择 "压力",获得了弱化H+-ATPase变异菌株5M1、5M6和5B1。与出发菌株L5（对照）相比,各变异株的H+-ATPase活力分别下降51.3%、27.7%和34.3％。对变异株5M1、5M6和5B1的形态及其生理特性研究表明,变异株的细胞较菌株L5细长。L5的ATP含量为0.63×10-18mol/个,明显低于变异菌株;变异株5M6和5B1的ATP含量分别为1.18×10-18mol/个和1.38×10-18mol/个;变异株5M1的ATP含量最高,约为1.60×10-18mol/个。当各菌株在MRS培养基中37℃培养12h后,变异株表现出较高的谷氨酸到γ-氨基丁酸的转化率,其中变异株5M6最高,为16.73%,变异株5M1和5B1谷氨酸转化率居中,分别为15.6%和15.4%,出发菌株L5的谷氨酸转化率最低,为13.91%。与出发菌株L5相比,变异株5M6、5M1和5B1的H+-ATPase活性下降导致其耐酸性和耐盐性降低。     

重组人胰岛素及C肽的分离纯化

对基因工程构建的含重组人胰岛素和C肽基因的毕赤酵母工程菌进行诱导表达，工程菌用甲醇诱导72 h后，离心去除菌体，上清液经中空纤维膜超滤后SP-Sepharose Fast Flow阳离子交换柱层析，0~400 mmol/L NaCl梯度洗脱，得到纯度大于92％的重组人胰岛素原。经Lys-C酶和羧肽酶B酶切后，Sephadex G-25柱分离，得到了重组人胰岛素和C肽。     

Genetic analysis and gene mapping of a narrow leaf mutant in rice ( Oryza sativa L.)

A narrow leaf mutant was obtained after T-DNA transformation conducted on a rice variety Zhonghua 11. Several abnormal morphological characteristics, including semi-dwarf, delayed flowering time, narrow and inward rolling leaves, and lower seed-setting, were observed. The rate of net photosynthesis (under saturate light) of flag leaves in the mutant was significantly lower than that of the wild type. Moreover, the leaf transpiration rate and stomatal conductance in the mutant flag leaf were lower than those of the wild type at the grain filling stage. It was found that the mutant phenotype was not caused by the T-DNA insertion. Genetic analysis showed that the mutant was controlled by a single recessive gene, designated as nal3(t). A genetic linkage map was constructed using a large F₂ mapping population derived from a cross between nal3(t) and an indica variety Longtefu B with 6 polymorphic markers on chromosome 12 identified from 366 SSR markers by the BAS method. Gene nal3(t) was mapped between the markers RM7018 and RM3331. Fine mapping of nal3(t) locus was conducted with 22 newly developed STS markers based on the sequence diversity around the region harboring nal3(t) between Nipponbare and 93–11, and nal3(t) was finally mapped to a 136-kb region between the STS markers NS10 and RH12-8. Supported by National High Technology Research and Development Program of China (863 Program) (Grant No. 2006AA10A102), National Natural Science Foundation of China (Grant No. 30600349) and Natural Science Foundation of Zhejiang Province (Grant No. Y306149)     

Prime role for an insulin epitope in the development of type 1 diabetes in NOD mice

A fundamental question about the pathogenesis of spontaneous autoimmune diabetes is whether there are primary autoantigens. For type 1 diabetes it is clear that multiple islet molecules are the target of autoimmunity in man and animal models. It is not clear whether any of the target molecules are essential for the destruction of islet beta cells. Here we show that the proinsulin/insulin molecules have a sequence that is a primary target of the autoimmunity that causes diabetes of the non-obese diabetic (NOD) mouse. We created insulin 1 and insulin 2 gene knockouts combined with a mutated proinsulin transgene (in which residue 16 on the B chain was changed to alanine) in NOD mice. This mutation abrogated the T-cell stimulation of a series of the major insulin autoreactive NOD T-cell clones. Female mice with only the altered insulin did not develop insulin autoantibodies, insulitis or autoimmune diabetes, in contrast with mice containing at least one copy of the native insulin gene. We suggest that proinsulin is a primary autoantigen of the NOD mouse, and speculate that organ-restricted autoimmune disorders with marked major histocompatibility complex (MHC) restriction of disease are likely to have specific primary autoantigens.     

Genetic analysis and gene fine mapping of a rolling leaf mutant (<Emphasis Type="Italic">rl</Emphasis><Subscript><Emphasis Type="Italic">11(t)</Emphasis></Subscript>) in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The exploration of new genes controlling rice leaf shape is an important foundation for rice functional genomics and plant archi-tecture improvement. In the present study, we identified a rolling leaf mutant from indica variety Yuefeng B, named rl_11(t), which exhibited reduced plant height, rolling and narrow leaves. Leaves in rl_11(t) mutant showed abnormal number and morphology of veins compared with those in wild type plants. In addition, rl_11(t) mutant was less sensitive to the inhibitory effect of auxin than the wild type. Genetic analysis suggested that the mutant was controlled by a single recessive gene. Gene Rl_11(t) was initially mapped between SSR markers RM6089 and RM124 on chromosome 4. Thirty-two new STS markers around the Rl_11(t) region were developed for fine mapping. A physical map encompassing the Rl_11(t) locus was constructed and the target gene was finally delimited to a 31.6 kb window between STS4-25 and STS4-26 on BAC AL606645. This provides useful information for cloning of Rl_11(t) gene.