盐生盐杆菌含耐盐相关基因DNA片段的克隆

将盐生盐杆菌总DNA的BamHI不完全酶切片段与质粒pUC19连接成重组质粒,并以重组质粒转化E.coli JM101.经X-Gal-IPTG法筛选白色重组子,结合高盐平板检测,已经获得了3株比原受体菌的耐盐性提高30％－67％的转化子。重组质粒酶切电泳分析表明,被克隆的盐生盐杆菌含耐盐相关基因的DNA片段长度在1．0－3．5kb之间。     

扩展青霉碱性脂肪酶cDNA的克隆和表达

采用TRIzol试剂一步法所抽提的总RNA的D（260）／D（280）值为1.82,甲醛变性电泳呈现真核微生物所特有的28S rRNA和18S rRNA条带。根据扩展青霉所产碱性脂肪酶（Lip PE)N末端20个氨基酸残基序列和真核生物mRNA3‘端具有poly(A)等所提供的生物信息,采用RT－PCR技术和3‘－RACE法扩增了Lip PE成熟肽编码区和3‘非编码区的cDNA,直接将该PCR产物克隆至pUCm-T载体中。序列分析表明,该碱性脂肪酶含有258个氨基酸,其中保守的五肽序列为Gly-His-Ser-Leu-Gly。进一步采用Clot Tech公司的SMART^TM PCR cDNA文库构建试剂盒,扩增、克隆和测定了自转录起始点至编码区的cDNA片段,从而完成了Lip EP完整cDNA的分析测定。最后将编码完整脂肪酶蛋白的cDNA克隆至pGEX-5X-3表达载体中,在大肠杆菌BL21中进行IPTG诱导表达,SDS－PAGE检测结果表明,所表达的GST－Lip EP融合蛋白分子质量约为53ku;免疫印迹（Western Blotting)技术证明了所克隆的cDNA确为编码扩展青霉WMC20718脂肪酶的基因。     

植物甜蛋白thaumatin基因重组表达质粒的构建

利用NDA重组技术,将植物甜蛋白thaumatin基因克隆至植物表达载体pBI121中,成功地构建了重组植物表达质粒pBI121-th。     

Expression of human erythropoietin directed by mWAP promoter in mammary gland of transgenic mice

The present work has generated transgenic mice with a hybrid gene construct consisting of genomic sequences encodinghuman erythropoietin (hEPO) and governed by regulatory sequences of mousewhey acidic protein (mWAP). The construct proved effective by transient expression in lactating animal. After introducing hybrid gene construct into single-cell embryo via pronuclear microinjection, surviving embryo are reimplanted into pseudopregnant foster mother mouse. 58 mice of 86 generation zero mice obtained were identified to be positive by PCR-Southern blot and genomic DNA Southern blot methods. The integration rate is 67%.hEPO was expressed in the milk of 16 mice of 39 mice measured byhEPO ELISA kit The expression level gets over 15 μg/mL.     

Regulatory effect of cotton leaf curl virus AC2 on virion sense promoter

The AC2 gene of cotton leaf curl virus (CLCuV) was obtained by polymerase chain reaction (PCR) . The total DNA of the CLCuV infected tomato leaves was used as template, and the amplified DNA fragment was inserted into a cloning vector. Transient expression vectors were constructed by inserting the AC2 gene into downstream region of CaMV 35S promoter. These constructs were delivered into tobacco and cotton leaf cells for transient expression by particle bombardment. The results indicated that the virion sense promoter was activated by AC2 and its activity increased remarkably. However, the activity of transactivated virion sense promoter was still lower than that of the complementary sense promoter. The expression pattern of transactivated virion sense promoter was similar to that of the complementary sense promoter, namely with high activity in both mesophyll and vascular tissues. The possibility of application of AC2 in plant genetic manipulation was also explored.     

定点突变内皮抑素Zn2+的结合位点及突变基因的克隆表达

从人胚肝组织中提取总RNA, 以逆转录聚合酶链式反应（RT-PCR）法获得人内皮抑素编码序列, 采用定点突变技术将His2和His4双突变为Leu2和Val4. 将突变基因cDNA插入含有T7启动子的质粒pET-28b中构建表达质粒pMendo, 转化大肠杆菌BL21(DE3), 筛选表达菌株BL21-Mute, 表达菌株经IPTG诱导后以包涵体方式产生大量内皮抑素突变蛋白. SDS-PAGE分析表明, 表达的重组蛋白占菌株可溶性蛋白质的30%. 复性、 纯化的内皮抑素突变蛋白纯度达到98%, 失去抑制人脐静脉内皮细胞增殖的活性.