真核表达载体pEGFP-N2/AT2R的构建及其在原代血管平滑肌细胞的表达

目的体外构建携带血管紧张素Ⅱ2型受体(ANGⅡType 2 Receptor,AT2R)基因的增强型绿色荧光蛋白真核表达载体,并观察其在大鼠血管平滑肌细胞(vascular smooth muscle cells,VSMCs)中的表达。方法以pUHD-10.3/AT2R质粒为模板,PCR扩增AT2R基因的全长cDNA序列,再将其克隆入载体pEGFP-N2,构建其真核表达载体pEGFP-N2/AT2R。以基因转染技术,将AT2R导入原代VSMCs。倒置荧光显微镜观测转染后VSMCs生长变化及AT2R在其中的表达等情况。图像分析技术检测AT2R在VSMCs中的转染效率。Western blot检测转染AT2R基因的VSMCs表达其编码蛋白。RT-PCR法对AT2R基因修饰的VSMCs进行鉴定。结果成功构建AT2R基因的真核表达载体pEGFP-N2/AT2R,该真核载体能携带AT2R基因转染并有效表达于VSMCs,其转染效率约40%,RT-PCR及Western blot均可检测到AT2RmRNA及蛋白在血管平滑肌细胞中高表达。结论成功地将克隆到的AT2R基因克隆入pEGFP-N2载体中,并实现了AT2R基因在原代VSMCs的表达。     

应用极限稀释细胞克隆形成实验方法检测血清活力

目的通过4批次血清活力检验与结果分析,介绍和评价极限稀释（LD）细胞克隆形成实验方法用于血清活力测定的实用性与优缺点。方法将检验细胞作极限稀释,极限值为13,使克隆单位内平均13个细胞,然后加入不同浓度的检验血清组,每1个浓度为1个克隆组,每组50个左右克隆单位;克隆培养5～7d左右,计数克隆阴性单位数。应用泊松分布函数处理实验数据,确定血清活力等级。结果用LD细胞克隆形成实验方法比较准确地测出4批次血清的活力：FBS-1活力等于Ⅱ级,Ⅲ级〉FBS-2活力〉Ⅳ级,FBS-3等于Ⅲ级,FBS-4无活力。结论LD细胞克隆形成实验方法用于检验血清活力,能准确反映血清活力状况,有较大的优势。     

新广谱大肠杆菌噬菌体IME11的分离及鉴定

目的 分离广谱大肠杆菌噬菌体并对其进行种属的鉴定。方法 以不同种临床致病性大肠杆菌为指示菌,从未经处理的污水样品中分离噬菌体;采用蛋白酶K/SDS的方法提取噬菌体基因组,制备重组载体,将阳性重组质粒进行测序;将测序结果进行BLAST,推测该噬菌体种属分类位置。结果 分别以大肠杆菌E1 ～ E17共17种细菌作为指示菌,成功分离出一种广谱噬菌体,可裂解31株临床分离致病性大肠杆菌中的13株,并将其命名为IME11;由噬菌体基因组限制性酶切片段分析表明其遗传物质为dsDNA;将噬菌体IME11基因组的2个随机片段测序结果进行BLAST,推测其属于N4-like噬菌体属。结论 分离出了一种广谱噬菌体,在分类学上属于N4-like噬菌体属。     

Association of distal chromosome 2q with IDDM in Japanese subjects

Summary An insulin-dependent diabetes mellitus (IDDM)-susceptibility gene (IDDM13) has recently been mapped to a region of distal chromosome 2q, which is syntenic to the region of mouse chromosome 1 containing a murine susceptibility gene for IDDM, Idd5. To determine the contribution of this region to IDDM disease susceptibility further and to narrow the region for positional cloning of susceptibility genes, we have studied the association of distal chromosome 2q with IDDM in the genetically distinct Japanese population. A 137 mobility unit (mu) allele at D2S137 locus was significantly associated with IDDM (odds ratio 1.92, p = 0.0016). Other markers, D2S301 and D2S143, located in the same region were not associated with IDDM, indicating that IDDM13 is in linkage disequilibrium with D2S137, but not with D2S301 or D2S143. The association of D2S137 with IDDM was observed in patients lacking one of two high risk HLA alleles, DQB1 ^* 0303 and DQB1 ^* 0401, but not in patients with either of these alleles. The frequency of high risk HLA alleles was significantly lower in patients with the susceptible allele at D2S137, suggesting that IDDM13 contributes to IDDM susceptibility in subjects without high risk genotypes at IDDM1. Demonstration of allelic association of D2S137 with IDDM localizes IDDM13 in the close vicinity (< 2 centiMorgans) of D2S137, greatly facilitating fine structure mapping and positional cloning of IDDM13. [Diabetologia (1998) 41: 228–232] Received: 27 March 1997 and in revised form: 3 October 1997     

Ethical and social issues of embryonic stem cell technology

Therapeutic cloning is debated as a cure for a host of diseases in the developed world. The likely source for the materials for therapeutic cloning, human ova, would be poor women and women from the developing world. The ethics and potential social consequences inherent in this technology are fraught and encourage the com modification and abstraction of one of the fundamental conditions of human life.     

人Ⅱ型大麻受体真核表达体系构建及其在 HEK293细胞中的表达

目的：构建人Ⅱ型大麻受体（ hCB2）基因GV230真核表达质粒,并检测hCB2基因在HEK293细胞中的表达。方法利用人脑皮质细胞的总RNA为模板,RT-PCR获得cDNA,通过酶切、连接及测序鉴定正确后,再将目的片段插入真核表达载体GV230,构建重组表达质粒GV230-hCB2,阳性克隆用脂质体瞬时转染HEK293细胞。激光共聚焦扫描显微镜和Western blotting法检测hCB2基因表达产物在细胞的表达情况。结果扩增出hCB2基因片段,成功构建了重组表达质粒,并检测到目的蛋白在转染细胞中表达,观察到hCB2受体在胞膜分布和表达。结论成功构建GV230-hCB2质粒,该质粒在HEK293细胞中能表达hCB2蛋白,此为进一步研究hCB2生物学功能奠定了实验基础。     

猪囊尾蚴抗原基因GP50的表达及鉴定

目的为了寻找猪囊尾蚴病新的免疫学候选诊断分子,克隆猪囊尾蚴抗原GP50编码基因,并进行表达、鉴定。方法设计合成引物,用PCR法从猪囊尾蚴cDNA文库中扩增出猪囊尾蚴抗原GP50基因编码序列,将其克隆入pGEM-T载体,然后在真核表达载体pBKCMV中亚克隆,用IPTG诱导表达,SDS-PAGE和Westernblot观察表达结果。结果RT-PCR法扩增出一条大小约897bp的特异性片段,克隆质粒pGEM-GP50和真核表达质粒pBKCMV作BamHⅠ和XhoⅠ双酶切和以重组质粒为模板进行PCR扩增,均可获得一条与PCR产物一致的DNA片段。诱导表达后,经SDS-PAGE可见一条约36kDa大小的融合蛋白条带,Western blot结果显示其可与猪囊尾蚴病人血清起反应。结论本实验成功地克隆了猪囊尾蚴抗原GP50编码基因,并在原核细胞中进行了表达及鉴定,为进一步的免疫诊断研究奠定了基础。     

Rescue of Infectious Sindbis Virus by Yeast Spheroplast-Mammalian Cell Fusion

Sindbis virus (SINV), a positive-sense single stranded RNA virus that causes mild symptoms in humans, is transmitted by mosquito bites. SINV reverse genetics have many implications, not only in understanding alphavirus transmission, replication cycle, and virus-host interactions, but also in biotechnology and biomedical applications. The rescue of SINV infectious particles is usually achieved by transfecting susceptible cells (BHK-21) with SINV-infectious mRNA genomes generated from cDNA constructed via in vitro translation (IVT). That procedure is time consuming, costly, and relies heavily on reagent quality. Here, we constructed a novel infectious SINV cDNA construct that expresses its genomic RNA in yeast cells controlled by galactose induction. Using spheroplasts made from this yeast, we established a robust polyethylene glycol-mediated yeast: BHK-21 fusion protocol to rescue infectious SINV particles. Our approach is timesaving and utilizes common lab reagents for SINV rescue. It could be a useful tool for the rescue of large single strand RNA viruses, such as SARS-CoV-2.