DinB基因的超表达短期内可显著降低大肠杆菌的死亡率

 [目的]探讨大肠杆菌dinB基因在进化上的选择优势.[方法]应用竞争共培养和超表达的策略,测定共培养后含dinB基因的目的菌株和对照菌株的生长优势.第1组为含质粒pUC-dinB-rrnb的E.coli AB1157＋含质粒pUC-dinBR-rrnb的E.coli YG7207;第2组为含质粒pUC-dinB-rrnb的E..coli AB1157＋含质粒pUC-phoE-rrnb的E.coli YG7207;第3组为含质粒pUC-phoE-rrnb的E.coli AB1157＋含质粒pUC-dinBR-rrnb的E.coli YG7207;第4组为卡那霉素抗性（K^＋）菌株YG7207与无卡那霉素抗性菌株AB1157.[结果]第4组中,卡那霉素抗性菌株YG7207与无卡那霉素抗性菌株AB1157在培养物中的比例保持着良好的稳定.第1组和第2组混合培养中,K^＋抗性的菌株在培养物中的比例急剧下降,并很快接近或等于0;第3组培养物中,K^＋菌株（YG7207）的比例呈明显上升.[结论]超表达dinB基因的大肠杆菌菌株的生长优势不是由于对数期的生长速率提高所造成,对数后期细胞死亡率降低应该是超表达dinB基因的大肠杆菌菌株的生长优势的主要原因.     

分泌表达人IL-12基因重组卡介苗菌株的构建和筛选

目的 筛选获得分泌表达人白介素12(IL-12)的基因重组卡介苗菌株.方法 采用PCR反应从pORF-h IL-12载体中扩增得到人IL-12基因的完整序列,克隆入大肠杆菌-分枝杆菌(E.coli-BCG)穿梭载体pMV361中,构建含人IL-12基因的重组质粒rpMV-IL-12.将纯化的rpMV-IL-12电穿孔转化卡介苗(BCG),通过卡那霉素抗性筛选、基因组PCR方法进行初步鉴定,再经热休克诱导表达后,分别收集培养上清液和细菌沉淀,进行SDS-PAGE分析,筛选得到IL-12基因重组BCG(rBCG-12).结果 成功构建含人IL-12基因的大肠杆菌-分枝杆菌穿梭质粒rpMV-IL-12,测序结果与GenBank收录序列一致,未发生突变.rpMV-IL-12电穿孔转化BCG,经卡那霉素抗性筛选及基因组DNA的IL-12目的 片段PCR扩增筛选得到rBCG-12重组菌.rBCG-12热休克诱导表达后的SDS-PAGE电泳,从培养上清液中检测到相对分子质量为70×103的蛋白质条带,而细菌沉淀中未见到特异性条带.结论 分泌表达人IL-12蛋白的重组BCG菌株构建、筛选成功.     

牙龈卟啉单胞菌FtsZ降解试验模型-clpP基因缺失株的构建

目的 建立牙龈卟啉单胞菌分裂关键蛋白FtsZ （FtsZPg）体内降解试验模型,即构建蛋白酶编码基因clpP缺失的E.coli MG1655.方法 将一段两端含靶基因同源臂的PCR产物电转入L-阿拉伯糖诱导后表达λ噬菌体重组蛋白、具有较强重组能力、含质粒pKD46的菌株E.coli MG1655感受态细胞,以PCR产物中的氯霉素抗性基因替代靶基因.结果 利用氯霉素抗性平板筛选阳性重组体,得到E.coli MG1655的ClpP蛋白酶基因敲除突变株.通过琼脂糖凝胶电泳和DNA测序最终鉴定阳性clpP基因缺失株.结论 本试验成功构建了E.coli MG1655 clpP敲除株;该敲除株有望在探讨重要牙周致病菌牙龈卟啉单胞菌分裂关键蛋白FtsZPg与蛋白酶clpP的关系中发挥一定作用.     

表皮葡萄球菌luxS基因敲除重组质粒的构建

目的构建1个含有luxS基因上、下游片段、抗卡那霉素基因的重组克隆质粒,用以敲除表皮葡萄球菌luxS基因。方法检索GenBank获得luxS基因序列以设计引物,以生物膜阳性表皮葡萄球菌基因组DNA为模板,采用高保真PCR扩增得到包含luxS基因上游片段、完整的luxS基因和luxS基因下游片段的长片段DNA;以pEASYT4质粒为模板扩增得到抗卡那霉素基因,再用内侧引物分别扩增luxS基因上、下游序列,按luxS基因上游片段＋抗卡那霉素基因＋luxS基因下游片段的顺序重组连接,转化JMl09感受态细胞,通过卡那霉素筛选、酶切分析和PCR一测序验证luxS基因敲除的重组克隆载体。结果经酶切后电泳验证重组质粒中各目的片段插入无误,PCR检测结果证明重组质粒luxS基因缺失,测序结果显示碱基无错配,含目的基因的重组质粒菌株在含卡那霉素培养平板内正常生长,证明插入的抗卡那霉素基因表达良好。结论表皮葡萄球菌luxS基因敲除重组质粒构建成功,为后续luxS基因缺陷株的构建奠定了基础。     

抗生素持续作用下细菌传代次数对接合频率的影响

目的探究抗生素作用下细菌传代次数对接合频率的影响及其可能机制。方法将本实验室构建的携带庆大霉素(GM)抗性基因的质粒p UCP24T转入E.coli SM10λpir获得重组菌E.coli SM10λpir(p UCP24T)。将重组菌在含30μg/m L GM的琼脂平板上不断传代,每次挑取单个菌落传代,获得第50代和100代的E.coli SM10λpir(p UCP24T)。分别以铜绿假单胞菌PAO1、大肠埃希菌EC600和肺炎克雷伯菌A10为受体菌,第1、50、100代E.coli SM10λpir(p UCP24T)为供体菌进行接合实验,计算接合频率。采用微量肉汤稀释法检测第1、50、100代E.coli SM10λpir(p UCP24T)对GM的最低抑菌浓度(MIC)值。分别提取第1、50、100代E.coli SM10λpir(p UCP24T)的全基因组DNA,构建DNA文库并进行高通量测序,对测序结果进行生物信息学分析。结果成功获得重组菌E.coli SM10λpir(p UCP24T)。第50、100代E.coli SM10λpir(p UCP24T)与三种受体菌的接合频率较第1代均显著增高(P0.05),第100代菌株对GM的MIC值大于2 048μg/m L,较第1和第50代菌株对GM的MIC值1 024μg/m L升高。第1、50、100代E.coli SM10λpir(p UCP24T)重组菌中携带GM抗性基因的p UCP24T质粒的拷贝数分别为601.3、1 808.2及9 190.5,呈现增高的趋势。结论在抗生素持续作用下,E.coli SM10λpir(p UCP24T)通过增加耐药质粒拷贝数使接合频率及MIC值升高,以适应抗生素的持续作用。     

pTn5GFP转座质粒用于耐药鲍曼不动杆菌生物被膜相关基因的研究

【立论依据】 鲍曼不动杆菌(Acinetobacter baumannii,AB)是一种革兰氏阴性球杆菌,是引起院内感染的主要菌种之一,多重耐药和泛耐药AB菌株感染更是给临床治疗带来极大的困难。生物被膜形成能力与AB耐药以及致病性密切相关。寻找AB生物被膜形成的关键基因对于AB相关疾病的预防和控制具有重要意义。转座子是一段特殊的DNA片段,在转座酶的作用下,可随机插入细菌基因组,使插入基因失活,改变相关表型。应用转座子随机突变策略可以找出特定表型对应的基因,但是以传统的抗生素抗性基因作为报告基因显然不能满足对多重耐药菌株的研究。 【设计思路】 绿色荧光蛋白(GFP)因为不需要单独加入底物并且易于观察而被广泛用来标记生物分子或细胞。已有研究表明,GFP基因插入细菌的染色体上后,可以有效地对细菌进行标记。基于转座质粒以及GFP的特点,本研究拟构建具有Tn5转座子和GFP基因的pTn5GFP转座质粒,利用GFP作为报告基因,构建临床分离的多重耐药AB突变文库,进而筛选出AB生物被膜形成的关键基因,并对其功能进行研究。 【实验内容】 以pGFPmut3.1质粒为模板,扩增GFP基因,将其连接入含有Tn5转座子的pRL27质粒,构建pTn5GFP转座质粒。利用该质粒对多重耐药AB菌株进行转座子突变,构造突变文库。对突变株的生物被膜情况进行分析,确定转座子插入位点,找出生物被膜形成的关键基因,并对其功能进行研究。 【材料】 临床多重耐药AB两株,R005和R036,自北京大学人民医院获得。常用工具菌E.coli CC118λπ和E.coli DH5α。pRL27和pGFPmut3.1质粒由原实验室毕业研究生提供。 【可行性】 已成功构建了pTn5GFP转座质粒,该质粒在容纳菌株E.coli CC118λπ具有绿色荧光表型,并已通过预实验初步确定了两株多重耐药AB生物被膜形成能力和最适实验条件。 【创新性】 本研究将GFP报告基因的便捷性以及转座质粒的转座性结合起来,有效解决了因耐药性而无法对泛耐药菌株构建突变文库的问题,结果直观,易于筛选,可行性高。     

鼠疫耶尔森氏菌 YopD 抗原基因在大肠杆菌中的克隆表达

目的：利用 DNA 重组技术,在大肠杆菌中获得融合表达的鼠疫耶尔森氏菌 YopD 抗原基因。方法根据查找文献及基因比对选取了鼠疫菌重要功能蛋白-YopD 蛋白。利用分子克隆技术克隆后,在原核系统中进行表达。根据云南玉龙菌株（D106004）全基因组序列设计引物,PCR 扩增目的基因片段。采用 pET-32a（＋）作为表达载体,通过双酶切和连接反应,将目的基因片段定向插入载体中,构建重组表达质粒。IPTG 诱导,使重组质粒在其宿主菌 E ．coli BL21（DE3）中表达。结果在大肠杆菌中成功获得了融合表达蛋白,即重组 YopD 蛋白。结论以质粒 pET-32a（＋）作为表达载体,鼠疫菌重要功能蛋白 YopD 能够在大肠杆菌 E ．coli BL21（DE3）中稳定高效地表达,为鼠疫潜在诊断靶点及新型疫苗选择的可能性奠定了基础。     

外源性指导序列体外转换临床大肠杆菌氯霉素抗性表型的实验研究

目的 研究外源性指导序列(EGS)体外逆转临床分离大肠杆菌氯霉素抗性的能力。方法 构建针对氯霉素(Cm)乙酰转移酶(cat)并含卡那霉素(Km)抗性基因筛选指标的：EGS重组质粒以及只含Km抗性基因但不含EGS的对照质粒。采用CaCl2方法将重组质粒导入临床分离的耐Cm大肠杆菌株中。通过质粒抽提、菌落PCR鉴定EGS阳性克隆子,分光光度计A600检测导入EGS质粒后耐Cm菌株在液体培养基中的生长率以及KB法检测在固体培养基中的药物敏感试验,探讨EGS分子逆转耐药菌的逆转效应。结果 以pEGFP-CI-EGS cat1 cat2重组质粒对16株临床分离的Cma大肠杆菌供试菌进行转化试验,EGS转化子在含Km的培养基上筛选。其中4株转化菌在Cm100～200μg／ml的液体培养基中生长受抑;在Cm 100～200μg／ml的固体培养基中药敏试验对Cm敏感;转化菌质粒抽提检测出特异EGS条带;菌落PCR扩增鉴定存在EGS。表明16株供试菌中4株临床分离株获得了耐药菌型的表型转换、耐Cm的大肠杆菌临床分离株恢复了对Cm的敏感性。结论 EGS分子具有逆转临床耐药菌为敏感菌的能力。     

含产气荚膜梭菌肠毒素基因重组质粒pET-28a-CPE的构建和表达

目的 构建含全长产气荚膜梭菌肠毒素(CPE)基因的重组表达质粒pET-28a-CPE并进行原核表达.方法 培养并提取表达肠毒素的产气荚膜梭菌标准菌株64615的基因组DNA,PCR扩增出全长CPE基因片段并连接入pET-28a载体质粒中,构建重组原核表达质粒pET-28a-CPE,进行酶切及测序鉴定,并转化入感受态大肠杆菌(E.coli)BL21中,用IPTG诱导CPE表达.结果 复苏、培养成功产肠毒素的产气荚膜梭菌标准菌株64615,成功构建了表达质粒pET-28a-CPE,经酶切及测序鉴定结果 与设计的完全相符,成功用E.coil BL21进行了原核表达,目的 蛋白CPE占总表达蛋白45.37%.结论 本实验成功构建并表达了含CPE的重组质粒pET-28a-CPE,为进一步观察CPE时前列腺肿瘤等的生物学作用奠定了基础.     

提高抗地高辛人单链抗体在大肠杆菌分泌型表达的研究

目的:提高从半合成噬菌体抗体库中克隆的抗地高辛(Dig) 人单链抗体(ADA scFv)在大肠杆菌(E.coli)的分泌表达水平.方法:①通过PCR从表达质粒pHEN2-ADA scFv扩增ADA scFv基因并重组于质粒p3MH构建表达质粒p3MH-ADA scFv后,转化到E.coli XL1-blue及HB2151; ②ADA scFv分别在带有上述2种表达质粒的E.coli HB2151 以及带有表达质粒p3MH-ADA scFv的E.coli XL1-blue进行表达,通过ELISA分析培养基上清中的可溶性ADA scFv;③在培养基中加入蔗糖、甘氨酸及Triton X-100,通过ELISA分析培养基上清中的可溶性ADA scFv.结果:①ADA scFv 在含表达质粒p3MH-ADA scFv的E.coli HB2151 及XL1-blue的分泌型表达水平比pHEN2-E.coli HB2151 表达系统高约 5 倍;②在培养基中加入蔗糖,可使ADA scFv 在含有表达质粒p3MH-ADA scFv的E.coli HB2151 及XL1-blue表达系统的分泌型表达提高 5 倍多.结论:通过采用不同的载体-E.coli表达系统以及在培养基中加入不被代谢的蔗糖,可使ADA scFv的分泌型表达提高 25 倍多.     

APPLICATION OF LORNOXICAM TO PATIENT-CONTROLLED ANALGESIA IN PATIENTS UNDERGOING ABDOMINAL SURGERIES

FOR anesthesiologis s ,treatingpostoperativepainhas alwaysbeen a problem.Althoughopioidshave been provedtobe effective,theirsideeffectscouldnotbeignored.With thedevelopmentofscienceand pharmacology,many drugs with aspectsof satisfactoryanalgesicefficacyand couldbe welltoleratedby patientshave been developed.And lornoxicamisone of them, which isa non-steroidalanti-inflammatorydrug (NSAID ), with analgesic, anti-infl-ammatory,andantipyreticproperties.Itseliminationhalf-time(3 to 5 hours) isle…     

Acupuncture Treatment of Vomiting

CASE HISTORY A female patient, 46 years old, head of the foreign affairs department of a certain university in Beijing, paid her first visit on October 9, 2006, with the chief complaint of vomiting for one month. She got vomiting after meals in early September. Before that, she had discomfortable sensation in the stomach due to angry with others, but she didn't pay much attention. Later, it developed into vomiting after eating. After the vomiting, the discomfort would be relieved, but with slight hypodynamia. She was once diagnosed as having 'neurogenic vomiting'. Having taken some western and Chinese drugs, the above symptoms were a little bit improved, but she would have nausea upon eating and with regurgitation. Because of the fear for vomiting, she did not dare to have food intake, with body weight reduction of 6 kilos in one month.     

Qiu Baoguo''''s Experience in Treating Consumptive Syndromes after Radio-Chemotherapies

Radiotherapy and chemotherapy are the important modern medical therapies for malignant tumors,yet they can also bring about serious local and systemic toxic side reactions so to decrease the patient;'s life quality,manifested by a series of consumptive symptoms.Having engaged in the combined work of Chinese and western medicine for nearly 50 years,the research fellow Qiu Baoguo in Henan Provincial Academy of TCM has developed his unique views on the TCM study of consumptive syndromes.The author of this essay had once the fortune tO follow Dr.Qiu in clinic,and specially would like to introduce in the following Dr.Qiu's experience in treating consumptive syndromes after radio-chemotherapies for patients with malignant tumor.     

Insomnia Due to Deficiency of Both the Heart and Spleen Treated by Acupuncture-Moxibustion and Chinese Tuina

OBJECTIVE: To observe therapeutic effects of the comprehensive therapy of acupuncture-moxibustion and Chinese Tuina for treatment of insomnia due to deficiency of both the heart and spleen. METHODS: 92 cases were divided randomly into the treatment group (treated by acupuncture-moxibustion and Chinese Tuina) and the control group (treated by acupuncture-moxibustion). RESULTS: The therapeutic effect of the treatment group was obviously superior to that of the control group (the CHI2 test showed P < 0.01). CONCLUSIONS: The comprehensive therapy of acupuncture-moxibustion and Chinese Tuina can give marked therapeutic effects for treatment of insomnia due to deficiency of both the heart and spleen.     

Analysis of CK-19,PDX-1,Nestin,Ngn3 in different donor islet purity in rats

Objective: To investigate if there are the CK-19, PDX-1, Nestin, Ngn3 positive cells in the donor islets of different purity in rats. Methods: Thirty male adult SD rats were randomly divided into 3 groups. Islets were isolated using digestion by ductal injection of collagenase. Group Ⅰ (n=10): Separating cell preparations were not purified, Group Ⅱ(n=10): Islet sediment was purified with 25% Ficoll400 ,Group Ⅲ (n=10): Islet sediment was purified with 25% and 11% Ficoll-400. The levels of protein of CK-19, PDX-1, Nestin and Ngn3 were detected by immunohistochemistry and the mRNA of CK-19, PDX-1, Nestin, Ngn3 was amplified by RT-PCR. Results: After two different purification methods applied, three islet preparations of different purities were obtained. The difference of islet purity was significant among various groups (P＜0.05). Compared with group Ⅱ and group Ⅲ,the protein and mRNA of CK-19, PDX-1, Nestin,Ngn3 were both higher in group Ⅰ; group Ⅲ was poorly expressed. Conclusions: The three different islet purity donor islet have different CK-19, PDX-1, Nestin, Ngn3 positive cells within them, indicating that there are some islet stem cells in the purified donor islet.     

Dr.Zhang Ren''''s Experience in the Acupuncture Treatment of Different Diseases with the Same Therapeutic Principle

Dr.Zhang Ren,the chief physician,is the chairman of Shanghai Acupuncture and Moxibustion Association.Having been engaged in medicine for about 40 years,he is experienced in treating various intractable diseases.In his long years of clinical practice,he advocates taking the TCM differentiation as the basis to seek for the acupuncture method for treatment of modern intractable diseases.The author of this essay had the fortune to follow Dr.Zhang in study.The following is a summary of Dr.Zhang's experience in the acupuncture treatment for different intractable diseases with the same therapeutic principle.     

Luo Lingjie''''s Experience in Treating Chronic Nephropathy

In treating chronic nephropathy,Luo Lingjie,a chief physician,pays attention to regulating the balance between yin and yang,treating infection if present,and removing pathogenic factors.He prescribes gentle drugs and uses carefully strongly warming-tonifying ones,emphasizes the importance of persuading the patient to persist in treatment with medication and nurse one's health for recuperation,and is good at combined use of TCM and western medicine therapy and brings the merits of various therapies into full play,with obvious theraoeutic effects.     

Acupuncture Combined with Spinal Tui Na for Treatment of Primary Dysmenorrhea in 30 Cases

Objective: To observe the therapeutic effects in acupunture treatment of primary dysmenorrhea combined with spinal Tui Na, and study its mechanism. Methods: Thirty cases of the treatment group were treated by acupuncture combined with spinal Tui Na, and thirty cases in the control group were treated by routine acupuncture. Results: The total effective rate was 93.3% in the treatment group, and 73.3% in the control group, with a significant difference between the two groups (P<0.05). Conclusions: Acupuncture combined with spinal Tui Na has good prospects for treatment of primary dysmenorrhea.