诺瓦克病毒广州株N蛋白基因的克隆、表达及抗原性鉴定

目的 克隆、表达和鉴定诺瓦克病毒广州株NVgz0l全长N蛋白.方法 在成功构建诺瓦克病毒广州株全基因组克隆并测序的基础上,将全长N蛋白基因克隆到表达载体pET28a( )上,经IPTG诱导表达后,利用Ni2 亲和层析柱对重组N蛋白进行纯化,并用Westem blotting和ELISA方法检测其抗原性.结果 重组诺瓦克病毒N蛋白基因在大肠杆菌中可以高效表达,其相对分子质量为62×103,可在Ni2 亲和层析柱上高度纯化;经抗原检测试剂盒检测和免疫学方法分析,均表明重组N蛋白具有良好的抗原性.结论 本研究克隆和表达了诺瓦克病毒广州株的全长N蛋白,为诺瓦克病毒诊断试剂和疫苗的开发等进一步的研究奠定了基础.     

小鼠CD36基因片段合成、表达及GST融合蛋白的表达和纯化

目的获取小鼠CD36基因片段,并高效表达和纯化GST-CD36融合蛋白。方法设计合成CD36的基因功能片段,测序后,通过酶切克隆至表达载体pGEX-5X-2,构建重组表达载体,并导入E.coli BL21宿主菌中,异丙基硫代半乳糖苷(IPTG)诱导表达重组的GST融合蛋白,亲和层析纯化表达产物,SDS-PAGE进行分析鉴定。结果获得小鼠CD36基因片段,测序结果与GenBank的基因序列一致,重组GST融合蛋白经SDS-PAGE分析,在相对分子量28.5kDa处,出现特异性蛋白条带,重组蛋白经GST亲和层析柱纯化后,得到了高纯度的融合蛋白。结论合成小鼠CD36基因片段,并在E.coli BL21中高效表达,亲和层析后获得高纯度GST-CD36融合蛋白。     

重组sCR1在毕赤酵母细胞中的表达、纯化及鉴定

目的:酵母细胞SMD 1168表达人sCR1,并对重组蛋白进行纯化。方法:从人外周血中提取总RNA,应用RT-PCR获得人sCR1全长cDNA,将其克隆入真核表达载体pPIC9K,构建含人sCR1的重组质粒(pPIC9K-sCR1);经测序鉴定正确后,将重组质粒转化入毕赤酵母菌细胞SMD 1168中,经甲醇诱导,表达产物经SDS-PAGE分析及W estern b lot鉴定,并通过N i2+-NTA agarose亲和层析纯化。结果:经甲醇诱导的含pPIC9K-sCR1的酵母细胞表达出重组人sCR1的融合蛋白,48~72 h sCR1融合蛋白表达量最高。此蛋白在凝胶上表现为M r大于31 KDa的蛋白区带,在W estern b lot分析中可被sCR1的CD35单克隆抗体(mAb)识别。经N i2+-NTA agarose亲和层析纯化后得到较纯的sCR1融合蛋白。结论:人sCR1融合蛋白在酵母细胞表达系统中的高水平表达,并且有与人体天然蛋白相同的抗原活性。     

SARS冠状病毒E蛋白的表达与纯化

目的研究SARS冠状病毒E蛋白对SARS诊断和预防的价值，利用原核表达系统克隆和表达E蛋白并纯化。方法采用RT—PCR从SARS冠状病毒RNA中扩增出编码N蛋白的基因；经克隆和测序分析后，亚克隆至表达载体DGEX-4T-2，转化大肠杆菌BL21（DE3），PCR和双酶切鉴定；阳性菌株经IPTG诱导，SDS—PAGE分析，进一步以SARS病人血清进行免疫印迹分析；大量诱导表达N蛋白，亲和层析予以纯化。结果RT—PCR扩增出E蛋白基因的特异片段，获得的阳性克隆序列与GenBank中登录的SARS冠状病毒的E蛋白基因序列同源性为100％；E蛋白基因被亚克隆至表达载体pGEX-4T-2，在BL21中获得表达，表达产物能被SARS病人血清识别。表达的E蛋白经亲和层析获得纯化。结论成功构建了SARS冠状病毒E蛋白的重组表达质粒，在大肠杆菌中表达的E蛋白的融合蛋白具免疫活性。     

大鼠IgE依赖组胺释放因子重组蛋白纯化效果的优化

目的提高大鼠IgE依赖组胺释放因子(rHRF)的可溶性表达水平,改善其亲和层析纯化效果,为深入研究该重组蛋白的生物学功能奠定基础。方法选择多克隆酶切位点BamHⅠ和XhoⅠ,将rHRF完整编码区基因从原有的pET-30a-rHRF重组质粒亚克隆至pET-28a载体,构建重组质粒pET-28a-rHRF;转化大肠杆菌BL21(DE3)后,通过改变IPTG浓度(0.1～1.0mmol/L),调节诱导表达温度(25～37℃)和时间(1～5h),筛选能够提高rHRF可溶性表达水平的条件;同时改进亲和层析纯化的条件,提高纯化效果。结果成功构建重组质粒pET-28a-rHRF,不同IPTG浓度、诱导温度和时间对重组蛋白的可溶性表达水平无明显影响。但是,与pET-30a-rHRF转化菌相比,pET-28a-rHRF转化菌所表达的重组蛋白因其N端和C端均带有6×his标签,增强了目的蛋白与树脂的结合力,而使亲和层析纯化效果(重组蛋白的浓度和纯度)显著提高。结论rHRF重组蛋白的可溶性表达水平不受IPTG浓度、诱导表达温度和时间的影响,但是可溶性重组蛋白两端均携带6×his标签可使亲和层析纯化效果显著提高,从而有利于获得足量rHRF用于进一步的生物学功能研究。     

严重急性呼吸综合征冠状病毒2核衣壳蛋白原核表达、纯化及其抗血清制备

目的 表达并纯化重组的严重急性呼吸综合征冠状病毒2（SARS-CoV-2）核衣壳（N）蛋白,通过免疫小鼠制备SARS-CoV-2 N蛋白抗血清。方法 将含有SARS-CoV-2 N基因的pET28a-N原核表达质粒转化大肠杆菌BL21（DE3）,用异丙基-β-D-硫代半乳糖苷诱导蛋白表达,用Ni-NTA亲和层析柱纯化SARS-CoV-2 N重组蛋白;将SARS-CoV-2 N重组蛋白联合锰佐剂通过肌内和皮下多点注射免疫BALB/c小鼠,获得抗血清;用蛋白质印迹分析检测SARS-CoV-2 N重组蛋白与SARS-CoV-2 N单克隆抗体及严重急性呼吸综合征冠状病毒（SARS-CoV）N多克隆抗体的反应;用间接免疫荧光实验检测小鼠抗血清与真核表达质粒转染细胞中SARS-CoV-2 N重组蛋白的反应。结果 成功诱导大肠杆菌表达出可溶性的SARS-CoV-2 N重组蛋白,相对分子质量约为55 000,纯化出该重组蛋白;蛋白质印迹分析结果显示SARS-CoV-2 N重组蛋白能与SARS-CoV-2 N单克隆抗体及SARS-CoV N多克隆抗体结合;间接免疫荧光实验检测结果表明制备的小鼠抗血清能与哺乳动物细胞中表达的SARS-CoV-2 N重组蛋白结合。结论 成功表达、纯化出SARS-CoV-2 N重组蛋白,并制备出该蛋白的小鼠抗血清,为后续建立SARS-CoV-2的快速诊断方法及开展SARS-CoV-2 N蛋白的功能研究奠定了基础。     

多功能转录因子CTCF重组质粒构建及其表达鉴定

目的 构建人CTCF cDNA全长及N端、Zn指、C端3个片段的原核融合表达载体,纯化融合蛋白并进行鉴定.方法 以编码CTCF全长序列的质粒为PCR模板,分别构建GST融合表达重组质粒pGEX-4T-2-CTCF,pGEX-4T-2-CTCF-N,pGEX-4T-2-CTCF-Zn,pGEX-4T-2-CTCF-C,转化大肠杆菌BL21,酶切、测序鉴定;优化IPTG诱导表达条件,并对亲和层析纯化的GST融合蛋白CTCF,CTCF-N,CTCF-Zn,CTCF进行Far-West-ern blot鉴定.结果 重组质粒经双酶切和测序鉴定证实构建成功.GST融合蛋白CTCF,CTCF-N,CTCF-Zn,CTCF-C均在大肠杆菌中成功诱导表达,融合蛋白经亲和层析纯化获得纯化蛋白.各纯化蛋白经SDS-PAGE和Far-Western blot鉴定,均为诱导表达蛋白质.结论 成功构建了GST融合表达CTCF,CTCF-N,CTCF-Zn,CTCF-C的重组质粒,对融合蛋白表达条件进行了优化.获得了高效表达的GST融合蛋白.     

弓形虫主要表面抗原1截短型片段的纯化与鉴定

目的:表达和纯化弓形虫SAG1,分析其免疫原性。方法:将诱导表达SAG1后菌体裂解,离心,分别收集其上清和沉淀;亲和层析分离纯化裂解上清液中的SAG1,SDS-PAGE和Western blot鉴定SAG1纯度和免疫原性。结果:诱导后重组体pGEX-SAG1/Bl21超声裂解上清液、8M脲溶解沉淀的上清中都含有融合蛋白GST-SAG1,从超声裂解上清液中纯化获得融合蛋白GST-SAG1。结论:亲和层析获得具有免疫原性的SAG1。     

梅毒螺旋体TpN17抗原的表达及纯化

目的:在大肠杆菌中重组表达梅毒螺旋体Tp N17抗原,通过亲和层析方法进行纯化,获得纯度较高的Tp N17抗原,利用该抗原建立梅毒诊断血清学方法。方法:将化学合成的Tp N17基因序列,克隆到p ET 22b质粒并转化到大肠杆菌BL21菌株中进行IPTG的诱导表达。利用Ni2+亲和层析柱对表达抗原进行纯化。结果:对插入片段进行测序,证实该序列结果与合成序列一致,并在大肠杆菌中成功表达Tp N17抗原,通过亲和层析获得纯度较高的目的抗原。结论:本研究中构建的大肠杆菌重组表达载体可有效的表达Tp N17抗原,经纯化后的抗原,可有望进一步应用于梅毒感染的血清学检测试剂盒的研发。     

小鼠TSR2-3/TSP-1基因片段的克隆及其GST融合蛋白的表达和纯化

目的：获取小鼠TSR2—3／TSP-1基因片段，并高效表达和纯化GST—TSR2—3融合蛋白。方法：应用RT—PCR技术，从小鼠肺总RNA中，获得编码TSR2—3的基因片功能片段，测序后，通过酶切克隆至表达载体pGEX-5X-2，构建重组表达载体，并导入E.coli BL21宿主菌中，IPTG诱导表达重组的GST融合蛋白，亲和层析纯化表达产物，SDS—PAGE和Westem blot进行分析鉴定。结果：获得小鼠TSR2-3基因片段，测序结果与GenBank的基因序列一致，重组GST融合蛋白经SDS-PAGE和Westem blot分析，在相对分子量39kDa处，出现特异性蛋白条带，重组蛋白经GST亲和层析柱纯化后，得到了高纯度的融合蛋白。结论：成功克隆小鼠TSR2—3基因片段，并在Ecoli BL21中高效表达，亲和层析后获得高纯度GST—TSR2—3融合蛋白。     

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.