大鼠肺表面活性蛋白C基因的克隆及原核表达

目的构建高氧下早产大鼠肺表面活性蛋白C基因原核表达质粒,实现其在大肠杆菌中的表达。方法孕21 d SD早产大鼠,生后12 h暴露于85%高氧中,7 d后处死,取肺组织,提取RNA,并合成cDNA,进行PCR扩增,克隆进pMD18-T载体,经过酶切和测序验证,成功构建原核表达载体pET-28a(+)-sp-c,重组质粒在大肠杆菌BL21菌株中经IPTG诱导表达,SDS-PAGE及Western blotting分析检测表达产物。结果经酶切和测序验证,成功构建pET-28a(+)-sp-c质粒,高效表达出相对分子质量21 000的融合蛋白。结论在大肠杆菌中成功表达SP-C重组融合蛋白,有助于研究SP-C蛋白与内质网之间的作用机制、SP-C错折叠蛋白在肺上皮A549细胞中的表达以及对AECⅡs增殖、分化及凋亡的影响。     

叶酸结合蛋白1真核表达载体的构建及鉴定

目的 构建pcDNA3.1.叶酸结合蛋白1(Folbp1)基因真核表达载体,检测其在肺腺癌A549细胞中的表达.方法 采用反转录.PCR技术从肺腺癌 A549 细胞中扩增Folbp1 基因的完整编码框,将其克隆到真核表达载体 pcDNATM 3.1/myc.His B,脂质体转染肺腺癌 A549细胞,通过G418筛选,建立稳定转染的肺腺癌 A549细胞株,并利用Western blot法鉴定其表达.结果 PCR扩增Folbp1基因全长编码区,电泳显示其分子大小与823 bp预期值一致;连接产物pMD.Folbp1转化感受态细菌,经氨苄西林筛选,提取质粒鉴定表明插入pMD.18T载体的目的 片段大小和方向正确;重组质粒扩增Folbp1 基因连入pcDNATM 3.1/myc.His B载体,酶切鉴定显示切出约5.1 kb符合载体大小的条带及符合插入片段大小的特异条带;转染pcDNA 3.1.Folbp1重组质粒的肺腺癌 A549细胞株经抗生素G418筛选,Western blot分析显示与预期大小一致的特异性条带.结论 pcDNA 3.1.Folbp1真核表达载体构建成功,并在肺腺癌 A549细胞内过表达,这将为研究Folbp1基因的功能及其肺发育中奠定了良好的基础.     

人肺表面活性相关蛋白A1在甲醇酵母的胞内表达

目的：研究人肺表面活性相关蛋白A1(SP-A1)在甲醇酵母中的胞内表达。方法：将编码正常人肺表面活性相关蛋白A1基因的cDNA克隆至甲醇酵母的胞内，表达载体pPIC3.5K，构建了重组质粒pPIC3.5K/SPA1，电穿孔方法转化至酵母宿主菌GS115和KM71，用PCR方法筛选阳性转化子，并用斑点印迹法、G418方法筛选多拷贝转化子，经甲醇诱导表达，SDS-PAGE和Western印迹法鉴定表达产物。结果：SP-A1基因在甲醇酵母中成功的表达了蛋白质，SDS-PAGE分析证实目的蛋白分子量为32 kD，可被SP-A多克隆抗体特异识别。结论：人组织特异SP-A1蛋白可在甲醇酵母中高效表达，表达产物具有免疫源性。     

恙虫病东方体Karp株相对分子质量为47蛋白基因的克隆与表达

目的了解恙虫病东方体Karp株相对分子质量为47蛋白的表达,检测其抗原性和免疫原性。方法采用PCR扩增恙虫病东方体Karp株相对分子质量为47蛋白成熟肽全基因序列并TA克隆,酶切PCR产物构建pGEX-47原核表达质粒,转化BL21,IPTG诱导表达相对分子质量为47重组蛋白,并检测其抗原性及免疫原性。结果1.扩增PCR产物约1300bp,测序结果与相对分子质量为47蛋白基因序列相同;2.构建原核表达质粒pGEX-47,纯化的重组蛋白SDS-PAGE分析在相对分子质量为47处,薄层扫描分析表达率为15.8%;3.Dot blot分析显示其有抗原性,小鼠免疫实验证实其有免疫原性。结论扩增相对分子质量为47蛋白的成熟肽全基因序列,在原核细胞得到表达,表达产物具有抗原性和免疫原性。     

鼠IL-10基因腺病毒载体的构建及在骨髓间充质干细胞中的表达

目的 构建携带鼠IL-10（rIL-10）基因重组腺病毒载体，并探讨其能否在鼠骨髓间充质干细胞（MSCs）中稳定表达。方法 rIL-10引物经PCR扩增rIL-10 cDNA序列，将回收到的656 bp rIL-10 DNA片段克隆入pcDNA3.1载体中，构建pcDNA3.1-IL-10，将其与腺病毒载体共转染HEK293细胞，进行细胞内同源重组，经测序及聚合酶链反应（PCR）鉴定重组是否成功；反复冻融裂解HEK293细胞，将获得的含rIL-10基因的病毒液感染MSCs，Western blot法检测rIL-10在MSCs中的表达。结果 经测序及PCR验证，rIL-10基因腺病毒载体构建成功，病毒滴度达4×10⁹ PFU/mL。含rIL-10基因的病毒液体外感染MSCs后，可检测到IL-10的表达。结论 构建重组腺病毒能够介导rIL-10基因在MSCs中的稳定表达，为下一步基因移植治疗炎症性肠病提供基础。     

人硫氧还蛋白1多克隆抗体制备及对内毒素血症新生大鼠的保护作用

目的 克隆人硫氧还蛋白1(hTrx-1)基因并在大肠杆菌表达体系中进行有效表达,制备其多克隆抗体.初步探讨hTrx-1对内毒素血症新生Sprague-Dawley(SD)大鼠的保护作用.方法 从人胎肝细胞中用RT-PCR的方法得到了hTrx-1全长基因,将它克隆到原核表达载体上,在大肠杆菌诱导表达并纯化,纯化的蛋白免疫...     

MicroRNA表达载体对大鼠KiSS1基因的干预效应

目的在细胞水平观察microRNA(miRNA)载体瞬时转染293T细胞株后对KiSS1基因转录、翻译的干预效应。方法以SD大鼠基因组DNA为模板,克隆KiSS1基因全长序列,构建重组载体pcDNA3.1(+)-KiSS1。应用miRNA设计软件,针对大鼠KiSS1基因的4个不同靶位点设计4对干扰序列及1对非相关性的阴性对照序列,得到miRNA表达载体p-KiSS1-miRNA1-4及p-KiSS1-miRNA-neg。利用脂质体介导将上述载体与pcDNA3.1(+)-KiSS1共转染293T细胞,采用荧光显微镜及免疫荧光技术检测转染72 h后细胞中各载体表达。采用实时荧光定量-PCR(RT-RCR)及Western blot技术观察转染48 h及72 h后miRNA表达载体对KiSS1基因的干预效应。结果荧光显微镜观察到miRNA载体转染293T细胞后可表达绿色荧光蛋白,免疫荧光结果显示转染pcDNA3.1(+)-KiSS1载体组可见红色的KiSS1蛋白在细胞质中表达;RT-PCR结果显示干预组细胞KiSS1 mRNA表达显著低于阳性对照组和阴性对照组(Pa<0.05)。其中p-KiSS1-miRNA2组KiSS1基因表达抑制最显著,降低了85%;Western blot结果显示p-KiSS1-miRNA2干预组KiSS1蛋白表达明显低于阳性对照组及阴性对照组。结论 4个miRNA载体对大鼠KiSS1基因均具有抑制效应,其中针对KiSS1 mRNA序列304～324 bp的p-KiSS1-miRNA2抑制效果最佳,为个体水平沉默KiSS1防治性早熟的研究提供依据。     

重组人类肥胖抑素在大肠杆菌中的大规模纯化

目的 探讨产率较高,高度纯化大肠杆菌表达重组肥胖抑素的方法,深入研究肥胖抑素的分子结构,生物活性及临床应用,对在大肠杆菌中表达的重组人类肥胖抑素进行纯化。方法 温度诱导表达携有人为肥胖基因（ｏｂ）片段的大肠杆菌重组子,采用酸沉淀结合凝胶过滤层析的方法从包涵体中纯化目的蛋白。结果 １周内从３Ｌ培养液（１０ｇ）湿菌体中可获得７０ｍｇ高度纯化的重组人类肥胖抑素。结论在短期内成功获得了高产高纯化的重组人类     

腺病毒介导多药耐药基因1保护骨髓的体外实验研究

目的构建表达多药耐药基因1（MDR1）的重组腺病毒载体，体外实验研究MDR1基因对骨髓的保护作用。方法用基因工程技术将MDR1的cDNA亚克隆至穿梭质粒pAdTrack-CMV上，利用PAdEasy系统进行细菌内同源重组，然后通过脂质体将正确重组体包裹并转染293细胞，并在其中包装、扩增、收获病毒。进一步采用PCR方法对重组腺病毒进行鉴定，利用穿梭质粒中所带绿色荧光蛋白GFP报告基因，对病毒滴度进行检测。将收集的重组腺病毒Ad5-MDR1感染小鼠单个核细胞，通过荧光显微镜、免疫组化及流式细胞仪对感染效率及保护作用进行检测，并用Westernblot法检测P-gp的表达。结果酶切鉴定及PCR结果证明多药耐药基因重组腺病毒载体构建成功，病毒滴度达8．3×10^11pfu／ml。对小鼠单个核细胞的感染效率可达10％～15％，转染小鼠单个核细胞48h后，有P-gp蛋白的明显表达。结论成功构建了表达MDR1的重组腺病毒载体，证实MDR转染对骨髓具有保护作用。     

LINE1-ORF1p过表达对肾母细胞瘤细胞WT_CLS1增殖的影响

目的 制备LINE1-ORF1p多克隆抗体，研究LINE1-ORF1p过表达对肾母细胞瘤细胞WT_CLS1增殖的影响。方法 利用基因工程方法原核表达LINE1-ORF1p，免疫家兔制备多克隆抗体。间接ELISA法检测抗体效价，通过Western blot及免疫组化方法检测抗体对LINE1-ORF1p的特异性识别能力。构建真核表达载体pEGFP-N1-LINE1-ORF1，转染WT_CLS1细胞，通过Western blot和qRT-PCR检测LINE1-ORF1蛋白和基因的表达情况，采用细胞增殖实验和平板克隆形成实验检测LINE1-ORF1p对WT_CLS1细胞增殖及肿瘤细胞克隆形成的影响。结果 制备的LINE1-ORF1p抗体效价 > 1：16 000，能对细胞及肿瘤组织内LINE1-ORF1p特异识别。转染pEGFP-N1-LINE1-ORF1的WT_CLS1细胞，其LINE1-ORF1的mRNA及蛋白水平显著增高（P < 0.05），细胞增殖能力和克隆形成能力都显著增强（P < 0.05）。结论 LINE1-ORF1p可以促进肾母细胞瘤细胞的生长和肿瘤细胞克隆形成，可能参与肾母细胞瘤的发病机制。     

Survivin在白血病中表达及其与CasPase、Fas相关性研究

目的　研究Survivin在白血病中表达的临床意义及其在白血病细胞凋亡中的作用。方法　应用蛋白印迹法检测1 8例白血病患者和 1 0例正常对照组患儿外周血中单个核细胞Survivin的表达 ;采用免疫组化法测定Fas和Caspase表达 ;运用四格表的确切概率法进行Survivin表达与白血病患者临床特点和Fas、Caspase表达的进行相关性分析。结果　 1 .白血病患者1 8例中 1 3例Survivin表达阳性 (72 .2 % ) ,正常对照组 1 0例Survivin检测均为阴性。Survivin表达在不同白血病类型、WBC组间有显著差异 (P <0 .0 5)。 2 .白血病患者 1 8例中 ,9例Fas表达阳性。经统计学分析 ,Survivin表达与Fas表达无相关性(P =0 .1 1 6)。 3 .白血病患者 1 8例中 5例Caspase 3表达阳性 ,两者比较 (P =0 .0 0 2 ) ,有显著性差异。结论　Survivin基因可选择性的在白血病患者外周血单个核细胞中表达 ,而在对照组中无Survivin表达 ,表明Survivin表达与白血病的发生有一定关系。Survivin表达情况可能成为白血病临床诊断、预后判断的有效指标。Survivin的作用位点可能不在Fas水平 ,而主要通过抑制Caspase 3表达 ,在凋亡途径的下游发挥抗凋亡作用。     

Expression,characterization, and biologic activity of recombinant human lactoferrin in rice

BACKGROUND: Lactoferrin has been suggested to have many biologic activities, such as facilitating iron absorption and having antimicrobial and antiinflammatory effects. In humans, several of these activities are likely to only be facilitated by human lactoferrin because they depend on the binding of human lactoferrin to specific receptors. Rice may be a useful vehicle to introduce recombinant human lactoferrin to infant foods because it has low allergenicity and is likely to be safer than using microorganisms or transgenic animals. METHODS: Recombinant human lactoferrin was expressed in the rice cell culture system, and its biologic activity was assessed by iron-binding and -releasing properties, antimicrobial activity, and binding and uptake to Caco-2 cells. The authors also compared the stability of recombinant and native human lactoferrins against heat, low pH, and in vitro digestion. RESULTS: Biologic activity of rice-expressed recombinant human lactoferrin was similar to that of native human lactoferrin. Heat-treated proteins retained their functional activities except with severe treatment at 100 degrees C for 8 seconds, which disturbed the iron-binding capacity of recombinant human lactoferrin. Both types of proteins retained their functional activities between pH 2 and 7.4. After in vitro digestion, 50% of both proteins were detectable by enzyme linked immunosorbent assay. The remaining native and recombinant lactoferrins retained antimicrobial and Caco-2 binding and uptake activities. CONCLUSIONS: The results indicate recombinant human lactoferrin has stability similar to native human lactoferrin when exposed to thermal treatment, pH treatment, and in vitro digestion, suggesting it may be active when added to infant formula.     

Cystic Neuroblastoma: Emphasis on Gene Expression, Morphology, and Pathogenesis

Cystic neuroblastoma (CN) is an unusual variant of neuroblastoma characterized by a grossly visible cyst(s) and almost always distinctive microcysts on light microscopy. Rarely, CN will appear solid grossly, but microcystification will be present. We examined the clinical, pathologic, and biologic features of 17 cases of CN. The majority of CN had been detected by prenatal ultrasound. The tumors were favorable stage, stroma-poor, but with low or intermediate mitotic-karyorhectic indices and had favorable biologic markers reflected by aneuploidy and by an absence of N-myc amplification and chromosome 1p deletions. However, the high trk expression typically identified in good risk tumors was absent. Although the complete natural history of CN is not fully defined, our experience suggests that some tumors progress in size, whereas others may spontaneously regress or mature. The clinical outcome is excellent, as is expected in localized and stage 4S neuroblastoma in infancy. Received June 13, 1996; accepted June 19, 1997     

An Introduction to Molecular Biology: Gene Structure, Expression, and Mutation

This mini-review outlines DNA structure and the regulation of gene expression. It then discusses the nature of mutations, techniques employed to detect them, and the limitations of these techniques.     

肾母细胞瘤N－myc、IGFⅡ基因mRNA的表达

应用Ｎｏｒｔｈｅｒｎｂｌｏｔ杂交，研究１３例肾母细胞瘤Ｎ－ｍｙｃ癌基因和类胰岛素生长因子Ⅱ（ＩＧＦⅡ）基因ｍＲＮＡ的表达，发现１２例Ｎ－ｍｙｃｍＲＮＡ的表达增强，１１例有明显的ＩＧＦⅡｍＲＮＡ的表达，分化较差的病，ＩＧＦⅡｍＲＮＡ多为高表达，提示肾母细胞瘤的发生发展过程中，可能也涉及癌基因和生长因子基因的异常。     

Expression, activity, and function of phosphodiesterases in the mature and immature ductus arteriosus

A patent ductus arteriosus is due in large part to increased sensitivity of the premature ductus to PGE2. After PGE2 stimulation, cAMP concentrations are higher in the immature than in the mature ductus. cAMP concentrations depend on the rates of adenyl cyclase production and phosphodiesterase (PDE)-mediated degradation. We used ductus from immature (n = 25) and mature (n = 21) fetal sheep to investigate whether a developmental increase in PDE activity could explain the diminished cAMP accumulation that follows PGE2 stimulation in the mature ductus. With advancing gestation, mRNA expression of the smooth muscle PDE isoforms (PDE1A, 1B, 1C, 3A, 3B, 4D, and 5A) increased in the ductus as did their hydrolytic activities. Selective inhibitors of PDE1, PDE3, and PDE4 relaxed the mature and immature ductus in the presence of inhibitors of prostaglandin and nitric oxide production. The mature ductus required higher concentrations of each of the PDE inhibitors to inhibit its tension to the same extent as in the immature ductus. There were no developmental changes in PDE expression in the fetal aorta. In conclusion, we observed a developmental increase in cAMP and cGMP PDE activity that contributes to the decreased sensitivity of the late-gestation ductus arteriosus to vasodilators like PGE2.     

Expression of adhesion molecules LFA-1, ICAM-1, CD44, and L-selectin in childhood non-Hodgkin lymphomas

BACKGROUND: The aim of the present study was to analyse the expression of adhesion molecules in childhood non-Hodgkin lymphomas and to correlate the findings with clinical features and prognosis. PROCEDURE: Samples were obtained from pleural and peritoneal fluids, bone marrow aspirates, and tissue biopsies from 21 patients (median age: 8 years). There were 9 T-cell and 12 B-cell lymphomas. The expression of CD18, CD44s, CD54, CD62L were investigated with flow cytometry by using monoclonal antibodies. RESULTS: Absence of CD18, which was independent from immunophenotype, was found in 67% of patients. Positive CD44s and CD62L expression were observed in 48 and 63% of the cases, respectively. In all of the cases with T-cell lymphoma, CD54 was negative, whereas 8 of 12 cases with B-cell lymphoma expressed this molecule (P = 0.005). There was no correlation between location of disease and the expression of adhesion molecules, except CD54 that was negative in all mediasten lymphoma (P = 0.004). CD62L (+) patients had more frequently stage IV disease than CD62L (-) ones (P = 0.01). Two-year overall survival was 83 and 29% in CD18 (+) and CD18 (-) cases; 55 and 36% in CD44s (+) and CD44s (-) cases; 46 and 42% in CD54 (+) and CD54 (-) cases; 42 and 50% in CD62L (+) and CD62L (-) cases. CONCLUSIONS: The expression of LFA-1 on lymphoblasts is lost in the majority of childhood non-Hodgkin lymphomas. ICAM-1 is not detected on neoplastic cells of patients with T-cell lymphoblastic lymphoma. L-selectin positivity correlates with disseminated disease. There is no significant relationship between the expression of adhesion molecules and the survival rates, although CD18(+) cases had better overall survival rate than CD18(-) cases.     

Congenital Sacrococcygeal Teratomas: Effect of Gestational Age on Size, Morphologic Pattern, Ploidy, p53, and ret Expression

Prognosis of infants born with sacrococcygeal teratomas (SCTs) correlates with gestational age (GA). The survival rate after 30 weeks of gestation is 75%, compared to 7% before 30 weeks of gestation. Studies correlating GA with size, morphologic composition of teratomas, ploidy or expression of cell cycle control proteins such as p53, and ret [a tyrosine kinase receptor of the GDNF (glial cell line–derived neurotrophic factors)] receptor family may provide information explaining differences in survival. Seven SCTs (GA 21 to 41 weeks), ranging in size from 5 to 15 cm, were evaluated for morphologic composition. DNA ploidy was assessed in mature and immature neural elements. Immunohistochemical reactivity with monoclonal antibodies recognizing p53, and ret was quantitated and correlated with morphological pattern and GA. Relative size of teratomas to infants' weight and content of immature neural tissues correlated inversely with advancement of GA. Yolk sac tumor (YST) and immature tissues showed aneuploid cell populations. Nuclear p53 reactivity was apparent in the teratoma with YST in the microcystic patterns, the neuroectodermal rosettes, and the glandular patterns. Ret reactivity was seen in osteoclasts adjacent to bone formation surrounding developing teeth in an immature teratoma, and in rare mature neural cells of one SCT of 35 weeks GA. The rapid growth of SCT (GA <30 weeks) correlates with increase in immature neural tissues. Our study confirms aneuploidy in YST and suggests aneuploid populations within immature tissues. p53 accumulates in a variety of patterns of YST and may be seen in immature components of SCTs. To understand the possible role of ret, further studies comparing ret expression in immature human tissues are needed. Received February 9, 1999; accepted August 17, 1999.