大肠杆菌Mn-SOD基因的克隆及表达

目的：实现Mn-SOD基因在大肠杆菌（E.coli）中的可溶性表达,并对表达产物进行活性测定。方法：用PCR方法从大肠杆菌2号基因组中扩增Mn-SOD基因编码区,克隆到pUCm-T Vector,测定核苷酸序列;再将基因编码区克隆到原核表达载体PET-28a,构建含Mn-SOD基因的重组表达质粒,转化到大肠杆菌BL21中进行IPTG诱导表达。结果：SDS-PAGE分析表明SOD的表达量约为细菌总蛋白的50%;黄嘌呤氧化酶法测定表达蛋白活性,菌体可溶性总蛋白中表达产物酶比活为3921.8 U/mg,是对照BL21的276.8倍。结论：Mn-SOD基因可在BL21中成功表达,产物具有高可溶性、高活性的特点。     

脱氢奎尼酸合成酶和奎尼酸脱氢酶基因在大肠杆菌中的共表达

目的：构建共表达载体pETDuet-aroB-qutB，在大肠杆菌中同时表达奎尼酸合成途径中的关键酶脱氢奎尼酸合成酶（DHQase）和奎尼酸脱氢酶（QDHase）。方法：分别以大肠杆菌（B21）和构巢曲霉（ATCC24919）基因组为模板，采用PCR法扩增得到脱氢奎尼酸合成酶基因aroB和奎尼酸脱氢酶基因qutB，克隆接入pETDuet-1载体中，转化入大肠杆菌BL21，在宿主菌中共表达脱氢奎尼酸合成酶和奎尼酸脱氢酶。结果：获得了包含pETDuet-aroB-qutB共表达载体的重组大肠杆菌。IPTG（0．5mmol／L）诱导重组菌4h后，脱氢奎尼酸合成酶和奎尼酸脱氢酶表达量分别占菌体总蛋白的18．7％和30．5％，酶活力为70．4U／L、40．2U／L，分别提高了14．1倍和22．3倍。结论：实现了脱氢奎尼酸合成酶和奎尼酸脱氢酶基因在大肠杆菌中的高效共表达，为基因工程法生物合成奎尼酸奠定了基础。     

人参Cu/Zn SOD原核可溶性表达条件的优化

目的在大肠杆菌中高效表达可溶性人参SOD,纯化后检测其活性。方法人参SOD cDNA插入到原核表达载体pET30α中,构建重组表达质粒,转化大肠杆菌BL21感受态细胞,IPTG诱导表达。分别对诱导时间、诱导温度、IPTG浓度以及离子浓度进行条件优化。对表达产物进行SDS-聚丙烯酰胺凝胶电泳和考马斯亮蓝染色检测。检测上清中蛋白的抗氧化活性。结果双酶切结果显示质粒构建成功,经过条件优化,表达的SOD蛋白主要存在于破碎菌体的上清中,表达量约占菌体上清总蛋白的50%,上清酶活达到363.9 U/mg。结论成功在大肠杆菌中高效表达了可溶性人参SOD蛋白,且具有较高的酶活性,为其工业化生产和应用奠定了基础。     

人血管生成抑制因子canstatin原核表达载体的构建及表达

目的：构建国人血管生成抑制因子canstatin的原核表达载体并在大肠杆菌BL21中表达canstatin重组蛋白。方法：用Trizol试剂提取人肝脏组织总RNA,通过RT—PCR扩增canstatin的cDNA,克隆到pMD18-T载体中并进行序列分析。将canstatin cDNA定向克隆于原核表达载体pET30a( )中,而后在大肠杆菌BL21中经IPTG诱导表达。结果：canstatin cDNA克隆人质粒pET30a( )获得了重组表达载体pET30a( )／Cans,canstatin的cDNA长度为684bp．编码227个氨基酸。IPTG诱导原核表达载体pET30a( )／Cans在大肠杆菌BL21中的表达量约占菌体总蛋白量的35％。结论：原核表达载体pET30a( )／hCans存大肠杆菌BL21中高效表达canstatin重组蛋白。     

乙醇对结核分枝杆菌Mr 38 000 蛋白在大肠杆菌中可溶性表达的促进作用

目的：探讨乙醇促进结核分枝杆菌Mr 38000蛋白的可溶性表达，并获得可溶性的Mr 38000重组蛋白．方法：采用聚合酶链反应(PCR)从结核分枝杆菌H37Rv基因组中扩增出Mr 38000蛋白的编码基因，用限制性内切酶消化后插入载体pGEx-4T-2中，将重组质粒转化大肠杆菌BL21。在乙醇添加剂存在下，经IPTG诱导，表达GST-38融合蛋白；聚丙烯酰胺凝胶电泳(SDS—PAGE)分析融合蛋白的相对分子质量及表达形式，免疫印迹法鉴定融合蛋白的活性．结果：经PCR扩增后证实为Mr 38000蛋白的基因，成功构建了具有正确基因序列的重组表达载体，在大肠杆菌BL21中诱导表达融合蛋白约占菌体总蛋白的18％．在乙醇存在下，可溶性目的蛋白的表达量约是无乙醇时的5倍．结论：在原核表达中乙醇作为一种添加剂可以促进结核分枝杆菌Mr 38000蛋白在大肠杆菌BL21中的可溶性表达．     

重叠PCR合成乙型肝炎病毒核心抗原基因及其在大肠杆菌中表达

目的：构建乙型肝炎病毒核心抗原（HBcAg）基因的融合表达质粒并在原核系统表达,为治疗性疫苗的研制奠定基础。方法：根据大肠杆菌偏爱密码子,利用Synthetic Gene Designer软件对HBcAg基因进行密码子优化,分成24条长约40bp寡核苷酸片段,将重叠PCR合成的HBcAg基因插入到pET30a中,经限制性核酸内切酶酶切鉴定和核酸序列分析无误后转化BL21（DE3）中,IPTG诱导表达,SDS-PAGE电泳分析表达情况。结果：酶切和测序鉴定表明,成功构建了重组质粒pET30a／rmcAg,SDS—PAGE分析显示目的蛋白以包涵体的形式在大肠杆菌中获得高表达,表达量占总菌体蛋白的64．3％。结论：应用重叠PCR合成HBcAg基因并在大肠杆菌中获得了高效表达。     

SZ—63单链抗体的基因构建、表达及免疫性研究

目的：基因构建与表达SZ-63单链抗体(SZ-63scFv)，降低单克隆抗体对人体的免疫源性。方法：运用基因工程手段通过一段小分子连接肽(Gly4Ser)3将SZ-63抗体的重链可变区基因和轻链可变区基因连接，构建成pET22b-63scFv表达载体，并导入大肠杆菌BL21（DE3）plys中进行表达，同时进行表达特性的研究。结果：pET22b-63scFv在BL21（DE）3plys中经IPTG诱导后，SZ-63scFv以包涵体形式存在，经复性处理后具有与纤维蛋白D—二聚体结合的活性，其表达量占菌体总蛋白的18％，SZ-63scFv具有13mg／g湿菌的表达量。结论：成功地获得了SZ-63scFv单链抗体并保留了与亲本抗体同一的抗体结合特性。     

质粒pGH/BL21在大肠杆菌中表达条件的研究

目的 寻找质粒pGH／BL21在大肠杆菌中的最佳表达条件。方法 当它在大肠杆菌中表达时，考查不同IPTG诱导浓度及不同的诱导时间对表达产物的影响，表达产物的量以SDS—PAGE电泳方法分析。结果 该基因的最佳表达条件为：IPTG诱导浓度1．0mmol／L，诱导时间4h。结论 IPTG对不同的基因在不同的表达体系中需不同的诱导条件，只有找到它的最佳诱导条件才能保证终产物的最大产率。     

HIV-1基质蛋白P17的原核表达与纯化

目的：HIV-1p17基因在大肠杆菌中高效表达并纯化目的蛋白。方法：①PCR法扩增的HIV-1p17基因片段克隆至pET28c质粒;②重组质粒分别在大肠杆菌BL21、BL21(DE3)、BL21(DE3)plysS及HMS174(DE3)中表达;③用Ni—NTA金属树脂层析法纯化目的蛋白;④用酶联免疫法和免疫印迹法检测纯化蛋白的特异性。结果：重组质粒在BL(DE3)中表达量最高,目的蛋白表达量占全菌体裂解物的32％,纯化后其纯度达95％,纯化蛋白可与p17McAb和HIV-1阳性血清发生特异反应。结论：带有HIV-1p17片段的重组质粒pET28c在大肠杆菌BL21(DE3)中获得高效表达,蛋白表达量可满足其免疫、生物活性的进一步研究;用Ni—NTA金属树脂层析法纯化蛋白,方法简便,蛋白丢失少,纯度高;纯化的重组蛋白HIV-1p17可用来临床早期诊断HIV-1感染者,同时可预测患者的临床进程。     

大肠杆菌核苷磷酸化酶的重组表达和活性

将大肠杆菌K-12菌株来源的嘌呤核苷磷酸化酶、尿苷磷酸化酶和胸苷磷酸化酶基因分别克隆到pET-11a载体并转化大肠杆菌BL21（DE3）宿主菌表达,通过SDS-PAGE分析和酶的活性测定,重组菌诱导表达后目的蛋白的表达量占菌体总蛋白的37%以上,与产核苷磷酸化酶的野生菌比较,酶的活性也有显著提高。在特异反应条件下,构建的工程菌可以用来高效催化核苷的转糖基反应。     

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.