The construction ofSchistosoma Japonicum vaccine BCG-Sj26GST and its identification

Summary The expression of foreign gene,Schistosoma Japonicum 26 ku antigen (Sj26GST),in Bacillus Calmette-Guerin (BCG),Mycobacterium CM. smegmatis) andEscherichia coli (E. coli) were studied. The cDNA fragment encoding Sj26GST was amplified by PCR using plasmid pGEX, which could express Sj26GST inE. coli as template. The Sj26GST cDNA was cloned into the downstream of humanM. tuberculosis heat shock protein (hsp) 70 promoter with correct reading frame, and then the DNA fragment containing hsp70 promoter and Sj26GST gene were subcloned together intoE. coli-Mycobacteria shuttle plasmid pBCG-2000 to construct the expression shuttle plasmid pBCG-Sj26. The recombinant BCG andM. smegmatis mc²155, which were electroplated with pBCG-Sj26, could express Sj26GST and the recombinantSchistosoma Japonicum vaccine BCG-Sj26GST was made. The recombinant Sj26GST (rSj26GST) were soluble and could be observed on SDS-PAGE at molecular weight of 26 ku. The content of rSj26GST accounted for 15% and 10% of total bacterial protein in BCG andM. smegmatis respectively. The results of Western blot showed the combination of rSj26GST with antibody of GST. This project was supported by Primary Foundation of China (No. 94-Y-19), Chinese National Nature Science Foundation (No. 339480022) and Chinese Health Foundation (No. 94-1-131).     

mGM-CSF重组质粒的构建、表达及活性鉴定

目的 构建pc—mGM—CSF重组质粒载体，为mGM—CSF基因治疗肿瘤的研究奠定基础。方法 采用RT—PCR方法从小鼠脾脏中获得目的基因mGM—CSF，克隆于pcDNA3.1／Myc-His(-)(A)质粒上，成为pc-mGM-CSF，用PCR、酶切进行鉴定，然后用脂质体转染小鼠骨髓瘤细胞SP2／0，用G418筛选后通过RT—PCR和SDS—PAGE鉴定，将转染SP2／0上清加入NFS-60细胞，检测蛋白活性。结果 重组质粒中含有mGM—CSF基因，在SP2／0中有表达，且表达产物能分泌到肿瘤细胞外，分泌到细胞外的产物用mGM—CSF依赖株NFS-60细胞检测证明具有生物学活性。结论 成功构建含mGM—CSF真核表达重组质粒，有助于进一步研究其抗肿瘤作用。     

人源性基质金属蛋白酶-1原核表达载体的构建与鉴定

目的构建人源性基质金属蛋白酶-1(MMP-1)原核表达载体. 方法从人肝组织提取总RNA,以此为模板,逆转录巢式 PCR扩增MMP-1全编码区基因片段,构建含目的片段的T载体克隆及原核表达载体pMAL-C2x重组质粒亚克隆,经双酶切及核苷酸测序进行分析鉴定. 结果成功地构建了人MMP-1原核表达载体. 结论构建的人MMP-1原核表达载体,为以后MMP-1融合蛋白的表达并获得具有抗原性的MMP-1融合蛋白奠定了基础.     

转粒细胞-巨噬细胞集落刺激因子基因真核表达质粒的构建及生物学活性鉴定

目的　构建小鼠转粒细胞 巨噬细胞集落刺激因子 (mGM CSF)真核表达质粒 pcDNA3 GM CSF ,转染红白血病细胞系FBL 3,并鉴定其活性。方法　采用分子克隆技术 ,构建mGM CSF真核表达质粒 pcDNA3 GM CSF ,电穿孔法将其导入红白血病细胞系FBL 3,G4 18筛选G4 18抗性细胞 ,限制性稀释法筛选G4 18抗性细胞克隆。PCR和RT PCR方法鉴定GM CSF基因整合和稳定表达。骨髓造血祖细胞增殖实验和骨髓造血祖细胞集落刺激实验鉴定其生物学活性。结果　采用PCR方法扩增mGM CSFcDNA序列 ,BamHⅠ和EcoRⅠ双酶切后装入 pcDNA3质粒 ,构建真核表达质粒 pcDNA3 GM CSF ,酶切和测序结果与预期相符 ,无插入、丢失、突变 ,方向正确。PCR和RT PCR结果显示 ,GM CSF基因整合到受体细胞染色体中并稳定表达。表达GM CSF的FBL 3 GM CSF细胞培养上清可明显刺激小鼠骨髓单个核细胞增殖 ,并能刺激干细胞形成克隆 ,形成克隆数为 (5 4 .6 7± 4 .83)个 ,形成率为 0 .5 4 7%。结论　构建mGM CSF真核表达质粒pcDNA3 GM CSF ,获得稳定表达该基因并具有生物学活性的细胞克隆 ,为制备转GM CSF基因瘤苗 ,探索白血病免疫治疗的可行性奠定基础     

鸭乙型肝炎病毒聚合酶结合模拟肽真核表达载体的构建

目的：构建与鸭乙型肝炎病毒聚合酶(DHBV P)特异性结合的模拟肽的重组真核表达质粒，为进一步将重组质粒转染原代培养鸭肝细胞研究模拟肽的作用奠定基础。方法：以噬菌体肽库中筛选出来的能特异性结合DHBV P的模拟肽基因为模板，扩增模拟肽基因片段，并应用基因重组技术构建其真核表达载体pEGFP-N1-模拟肽基因(pEGFP-N1-M)。结果：经鉴定，目的基因片段以正确的方向插入载体pEGFP-N1中，序列正确，对码正确。结论：成功构建了重组真核表达质粒pEGFP-N1-M。     

Influence of Humans on Evolution and Mobilization of Environmental Antibiotic Resistome

The clinical failure of antimicrobial drugs that were previously effective in controlling infectious disease is a tragedy of increasing magnitude that gravely affects human health. This resistance by pathogens is often the endpoint of an evolutionary process that began billions of years ago in non–disease-causing microorganisms. This environmental resistome, its mobilization, and the conditions that facilitate its entry into human pathogens are at the heart of the current public health crisis in antibiotic resistance. Understanding the origins, evolution, and mechanisms of transfer of resistance elements is vital to our ability to adequately address this public health issue.     

Overexpression of cytoglobin gene inhibits hypoxic injury to SH-SY5Y neuroblastoma cells

A plasmid for cytoglobin expression, pAcGFP1-C1-cytoglobin, was transfected into SH-SY5Y cells. Cobalt chloride was used to establish a model of hypoxia. Western blotting indicated that cytoglobin was overexpressed and there was low expression of hypoxia-inducible factor-1α in SH-SY5Y cells after transfection. Following cobalt chloride-induced hypoxia, cytoglobin and hypoxia-inducible fac-tor-1α expression gradually increased in SH-SY5Y cells. Flow cytometry showed that with increas-ing duration of hypoxia, the proportion of normal cells significantly diminished in the transfected and non-transfected groups. The proportion of cells in the early stages of apoptosis increased. However, the proportion of apoptotic cells was significantly lower in the transfected group compared with the non-transfected group. These results demonstrate that cytoglobin and hypoxia-inducible factor-1α are strongly up-regulated by hypoxia, and that there is a strong relationship between hy-poxia-inducible factor-1α and cytoglobin during hypoxic injury.     

Polymeric nano- and microparticle technologies for oral gene delivery

Gene therapy refers to local or systemic administration of a nucleic acid construct that can prevent, treat and even cure diseases by changing the expression of genes that are responsible for the pathological condition. Oral gene therapy has significant promise for treatment of local diseases such as inflammatory bowel disease and for systemic absorption of the expressed protein therapeutics. In addition, efficient oral delivery of DNA vaccines can have significant impact in disease prevention. The use of polymeric gene delivery vectors promises the translation of this experimental medical concept into clinical reality. This review addresses the challenges and opportunities in the development of polymer-based nano- and microparticle technologies for oral gene therapy. Specifically, the discussion is focused on different synthetic and natural polymers used for formulating nano- and microparticle technologies and the use of these delivery systems for oral DNA administration for therapeutic and vaccination purposes.