人乳头瘤病毒重组质粒的构建及蛋白表达

目的 构建重组原核表达质粒获得人乳头瘤病毒18型L1片段(HPVl8LI)活性蛋白。为进一步研制人乳头瘤病毒18型(HPV18)基因工程疫苗打下基础。方法 以重组质粒(pBR322-HPV18)为模板，利用PCR方法扩增HPV18L1DNA片段。将HPV18L1DNA与质粒(pUC19)重组构建重组质粒(pUC19-HPV18L1)。用酶切电泳验证重组结果的正确性；并通过测序检查质粒重组后序列有无变化。再利用质粒(pQE32)做表达载体构建重组质粒(pQE32-HPV18L1)，并用酶切电泳验证。将重组质粒pQE32-HPV18L1转入工程菌(M15)。用酶切电泳验证重组工程菌(pQE32-HPV18L1／M15)正确性。利用SDS-聚丙酰胺凝胶电泳(SDS-PAGE)检测HPV18L1目的蛋白。采用不同浓度异丙基-β-D-半乳糖苷(IPTG)、不同诱导时间来优化HPV18L1目的蛋白表达条件。利用Western印迹检测蛋白质(Western blot)鉴定HPV18L1目的蛋白的特异性。结果PCR扩增DNA片段约为1．7Kb。与预期结果相同。克隆重组质粒pUC19-HPV18L1酶切后显示的酶切图谱与预期相同。而且测序验证插入片段全序列无改变。表达重组质粒pQE32-HPVl8L1酶切图谱亦与预期相同。蛋白表达条件优化结果为1mmol／LIPTG诱导4h后，获得HPV18L1最佳蛋白表达量：SDS-PAGE显示，约63KD处可见目的蛋白带，与预期结果一致。Westem blot鉴定，在约63KD处可见目的条带，与预期结果一致。结论 成功构建表达重组质粒pQE32-HPV18L1并能表达HPV18L1目的蛋白。     

充血性心力衰竭患儿血清白细胞介素-18和淋巴细胞CD54变化

目的探讨IL-18和CDs4在小儿充血性心力衰竭（CFIF）发病中的作用，及其对z],JLCFIF的诊断价值。方法CFIF患儿52例，心功能按修订的Ross和Reithman评分系统。心功能Ⅱ级（3～6分）18例、Ⅲ级（7～9分）17例，Ⅳ级（10～12分）17例。原发疾病为扩张性心肌病、心内膜弹力纤维增生症及先天性心脏病等。对照组15例为上呼吸道感染患儿。血清IL-18水平用ELISA法进行测定，采用流式细胞术检测外周血淋巴细胞CDs4表达。结果CFIF患儿血清IL-18及CDs4均显著增高（P均〈0．001），且随CFIF加重渐增加，治疗后两者均较治疗前明显降低（P均〈0．05）；且两者在心肌病组、先天性心脏病组及其他病组3组间比较差异无显著性（P均〉0．05），但均显著高于对照组（P均〈0．01）。结论IL-18和CDs4可能参与小儿CFIF发生发展。可以作为评价CHF严重程度的生物化学标记；CFIF作为一种临床综合征，无论最初病因是否相同，免疫反应可能都具有某些共同特征。     

油酸-内毒素序贯致新生大鼠急性肺损伤IL-18水平的变化

目的:探讨油酸-内毒素序贯致伤新生大鼠的IL-18水平,在急性肺损伤中的作用。方法:采用油酸加内毒素脂多糖腹腔注射新生大鼠致肺损伤模型,在油酸致伤后4h实施内毒素第2次致伤,收集血浆检测IL-18水平,同时设立单剂量内毒素致伤组和生理盐水为对照组,每组12只。结果:油酸-内毒素致伤新生大鼠血浆IL-18水平均显著高于单剂量内毒素致伤组和生理盐水对照组(P<0.01)。结论:油酸-内毒素组IL-18水平显著高于单剂量内毒素致伤组和生理盐水对照组,提示致炎因子IL-18可能在急性肺损伤的发生和发展中起重要的作用。     

IL-18在大肠杆菌中的可溶性表达和纯化

目的：研究人IL-18成熟蛋白（mhIL-18）在大肠杆菌中的高效的非包涵体表达以及纯化方案。方法：利用PCR方法扩增出mhIL-18基因，构建融合型表达载体pPTYB1-IL18，转化入大肠杆菌ER2566中，IPTG诱导表达，优化表达条件，超声裂解细胞，采用SDS-PMGE和Weslern blot分析重组蛋白的表达，亲和层析后用MTT法检测表达mhIL-18的活性。结果：成功构建载体并转化入ER2566后，经IPTG诱导，表达量可占菌体总蛋白的26％以上，其中80％以上的mhIL-18融合蛋白以可溶、有活性的形式存在。亲和层析后的mhIL-18纯度可达95％，具有显著的IL-18的生物学活性。结论：成功的在大肠杆菌中的高效以非包涵体表达mhIL-18，并具有显著的IL-18的生物学活性。     

Long-term persistence of molecular disease after histological remission in low-grade gastric MALT lymphoma treated with H. pylori eradication. Lack of association with translocation t(11;18): a 10-year updated follow-up of a prospective study.

BACKGROUND: Localized low-grade gastric mucosa-associated lymphoid tissue (MALT) lymphoma can regress after Helicobacter pylori eradication, but IgV(H) gene monoclonality may persist. We studied the long-term histological and molecular follow-up of 24 patients and the possible association of t(11;18) with the persistent monoclonality. PATIENTS AND METHODS: From January 1994, 24 untreated patients with stage I low-grade gastric MALT lymphoma associated with H. pylori were prospectively studied. They all received eradication treatment and were sequentially followed-up with endoscopies for histological and molecular studies. Rearrangement of the IgV(H) gene was studied by PCR analysis. MALT1 locus alterations were studied by FISH. RESULTS: Twenty-two of the 24 patients (91%) achieved disappearance of the lymphoma. Eighteen (82%) of the 22 histologically cured patients and 16 of the 19 (84%) with long follow-up had monoclonality. Three patterns of development of IgV(H) gene rearrangements were observed: four patients (21%) had polyclonal rearrangements; eight (58%) had maintained/intermittent monoclonality and four (21%) had occasional monoclonality, mostly after H. pylori reinfection. Only one patient (6%) with persistent monoclonality relapsed. The remaining 18 patients maintained the remission, despite the persistent monoclonality in 15, for a median of 66 months (range 20-113). t(11;18) was not found in any of the patients with persistent monoclonality. Time and the number of endoscopies performed were not related with the occurrence of monoclonality. CONCLUSIONS: In stage I low-grade gastric MALT lymphoma eradication of H. pylori achieves prolonged histological remission in 90% of patients, but molecular remission is not accomplished in most cases. Molecular disease persists for years, but is not associated with t(11;18).     

Population study of Aymara Amerindians for the PCR-DNA polymorphisms HUMTH01, HUMVWA31A, D3S1358, D8S1179, D18S51, D19S253, YNZ22 and HLA-DQα

Allele and genotype frequencies for eight DNA polymorphisms (HUMTH01, HUMVWA31A, D3S1358, D8S1179, D18S51, D19S253, YNZ22 and HLA-DQalpha) were determined in a population sample of Aymara Indians from Bolivia using PCR. No deviations of the observed allelic frequencies from Hardy-Weinberg equilibrium were found for all the systems studied. Significant differences in the allele frequencies were found between the Aymara and Quechua populations only for HUMVWA31A, which suggests a certain degree of genetic differentiation between the two populations.     

New radiolabelling chemistry: synthesis of phosphorus–[18F]fluorine compounds

The feasibility of synthesizing compounds containing the P–¹⁸F bond has been demonstrated by labelling the pesticide, cholinesterase inhibitor Dimefox (N,N,N′N′‐tetramethylphosphorodiamidic fluoride) with F‐18. Radiolabelling was achieved in high radiochemical yield (96%) by nucleophilic substitution of the chloro group attached to phosphorus, in the oxidation state P(V), by ¹⁸F^? (activated with tetrabutylammonium carbonate in acetonitrile). Given the large number of important biological molecules possessing phosphorus such as oligonucleotides, phospholipids as well as phosphorylated proteins, sugars and steroids, this new labelling chemistry may provide an additional route to radiolabelling these biologically important compounds for use in PET. Copyright © 2005 John Wiley & Sons, Ltd.     

Synthesis of 2‐[(2‐chloro‐2′‐[18F]fluoroethyl)amino]‐2H‐1,3,2‐oxazaphosphorinane‐2‐oxide (18F‐fluorocyclophosphamide), a potential tracer for breast tumor prognostic imaging with PET

A fluorine‐18 labeled analog of the widely used chemotherapeutic agent cyclophosphamide was synthesized as a tracer for prognostic imaging with positron emission tomography. 2‐[(2‐Chloro‐2′‐[¹⁸F]fluoroethyl)amino]‐2H‐1,3,2‐oxazaphosphorinane‐2‐oxide (¹⁸F‐fluorocyclophosphamide), was prepared by direct halogen exchange reaction from the parent cyclophosphamide. In small‐scale syntheses, radiochemical yields of up to 4.9% and specific activities of 960 Ci/mmol were achieved in a total synthesis time of 60–75 min. The [¹⁸F]‐labeled cyclophosphamide analog with radioactive purity >99% and chemical purity >96% was suitable for in vivo (microPET imaging) and ex vivo studies of a murine model of human breast tumors. Copyright © 2005 John Wiley & Sons, Ltd.     

稽留流产患者血清和绒毛组织E_2、P、β-hCG、IL-18、PDCD-5的表达及其预警意义

目的:探讨E_2、P、β-hCG、IL-18、PDCD-5在稽留流产患者血清和绒毛组织的表达及其预警意义。方法:选取2018年1—6月我院收治稽留流产妇女30例作为实验组,同期就诊正常早孕妇女(要求终止妊娠)30例为对照组,测定患者血清和绒毛组织E_2、P、β-hCG、IL-18、PDCD-5表达水平,通过统计学软件分析这些指标与孕妇发生稽留流产的相关性。结果:与对照组相比,实验组受试者血清、绒毛组织中E_2、P和β-hCG水平明显较低(P<0. 05),IL-18和PDCD-5水平明显较高(P<0. 05)。Logistic多元回归分析结果显示,血清E_2、P、β-hCG低表达以及IL-18、PDCD-5高表达为孕妇发生稽留流产的危险因素(P<0. 05)。结论:血清、绒毛组织中E_2、P、β-hCG低表达以及IL-18、PDCD-5高表达与孕妇稽留流产发生有关,具有一定预警作用。