人源天然Fab噬菌体抗体库的构建及抗c-Met抗体的筛选?鉴定

目的:构建大容量人源天然Fab噬菌体抗体库,筛选抗c-Met特异性抗体并进行初步鉴定.方法:采集20位健康成人的骨髓淋巴细胞.用PCR扩增人Fab片断抗体基因,插入载体pComb3XSS内,构建人源天然Fab抗体库.以固相化的抗原对抗体库进行6轮筛选后,随机挑选60个克隆用Phage ELISA、BstO I酶切片断分析进行检测,阳性克隆作可溶性表达和鉴定.结果:构建的Fab噬菌体抗体库的库容为1.2×109,从中筛选到1株与c-Met特异性结合的人源抗体克隆,命名为AM2-26.DNA序列分析证明为人免疫球蛋白可变区基因,Western blot证实为人源抗c-Met Fab抗体片段,ELISA特异性鉴定阳性.结论:构建了大容量人源天然Fab抗体库,从中获得1株抗c-Met人源Fab抗体片段,有望为抗肿瘤药物的研制提供新的候选分子.     

大容量天然人源Fab抗体库的构建及日本血吸虫抗独特型抗体的筛选、鉴定

目的:构建大容量天然人源Fab抗体库,筛选全人源日本血吸虫抗独特型抗体并进行初步鉴定。方法:采集15位健康成人的骨髓淋巴细胞,构建天然人源Fab抗体库。并以日本血吸虫可溶性虫卵抗原(SEA)及成虫抗原(SWAP)免疫鼠血清IgG包板进行筛选,经ELISA鉴定后,对阳性克隆进行可溶性表达。结果:构建了2×109大容量天然人源Fab抗体库,经核苷酸序列分析,证实插入片段为Fab基因片断。经过6轮筛选后,从富集的次级抗体库中随机挑取80个克隆,用ELISA法鉴定得到4个可与免疫鼠血清(Ab1)特异性结合,而不与SEA及SWAP结合的单克隆抗体(C12、C19、D1、D2),其中C12经SDS-PAGE电泳显示插入片断正确。结论:成功构建了大容量天然人源Fab抗体库,并从中获得人源日本血吸虫抗独特型抗体C12,为研制用于人体血吸虫病免疫预防的抗独特型抗体疫苗奠定了基础。     

人源性抗核抗体Fab片段的筛选及鉴定

目的:制备人源性抗核抗体Fab片段.方法:通过4轮淘筛,富集已构建的人源性抗核抗体Fab片段噬菌体抗体库,间接ELISA法鉴定4轮淘筛后抗核抗体Fab片段噬菌体抗体;提取阳性克隆的噬菌粒DNA,切除gⅢ基因片段,自连接后转化大肠杆菌XL1-Blue,以IPTG诱导表达可溶性人源性抗核抗体Fab片段;应用间接ELISA法及荧光免疫法对表达上清进行鉴定.结果:第4轮洗脱的噬菌体滴度较第1轮增加200条倍,从抗核抗体Fab片段噬菌体库中筛选出2株阳性克隆,切除gⅢ基因后自连的噬菌粒DNA经XhoⅠ单酶切证实连接成功.间接ELISA法检测结果显示:制备的可溶性人源性抗核抗体Fab片段均呈现抗dsDNA阳性,具有抗原特异性;免疫荧光法结果显示:Hep2细胞和猴肝脏组织细胞核显示均质型荧光,绿蝇短膜虫的动基体显示均质型荧光.结论:成功制备具有抗原特异性的可溶性、人源性抗核抗体Fab片段,为高亲和力抗核抗体Fab片段的制备奠定了基础.     

人源全分子抗c-Met 抗体的制备及对鼻咽癌细胞生物学特性的影响

目的:利用基因工程技术制备全分子人源抗 c-Met抗体,分析该抗体免疫学特性,并观察该全分子抗体对鼻咽癌细胞增殖?迁移和侵袭的影响?方法:设计引物扩增抗c-Met Fab抗体的重轻链可变区序列,将其分别克隆到真核表达载体pFUSE-CHIg-hG1 和 pFUSE-CLIg-hκ 中?转染真核细胞,表达产物用Protein A柱纯化?通过ELISA?Western blot和免疫荧光等方法鉴定该抗体的免疫学特性?CCK-8检测抗体对人鼻咽癌细胞增殖的抑制作用,划痕实验?Transwell侵袭实验分析抗体对人鼻咽癌细胞迁移和侵袭的影响?结果:成功重组全分子人源抗c-Met 抗体的表达载体,获得的全分子抗体保留了与抗原的特异性结合活性?CCK-8实验中结果显示全分子抗体能抑制c-Met阳性鼻咽癌细胞CNE2和HONE1的增殖?细胞划痕和Transwell侵袭实验结果显示全分子抗体能抑制c-Met阳性细胞CNE2和HONE1的迁移和侵袭能力?结论:本研究成功制备人源全分子抗c-Met抗体,该抗体有明显的中和活性,为探索鼻咽癌分子靶向治疗奠定了基础?     

从大容量噬菌体抗体库中筛选人源性抗角蛋白scFv

目的从大容量噬菌体抗体库中筛选人源性抗角蛋白单链抗体(scFv),测定其特异性结合活性并进行基因序列分析。方法以混合分子量人表皮角蛋白包被固相,对构建的大容量噬菌体抗体库进行筛选,经4轮“吸附洗脱扩增”后,挑取单克隆,用ELISA法测定特异性结合活性,并对阳性克隆抗体基因进行DNA指纹分析及序列同源性分析。结果经4轮筛选,获得9株可表达抗人角蛋白单链抗体的克隆,酶切鉴定正确后,经DNA指纹分析及序列同源性分析证明为不同的抗体基因。结论利用噬菌体抗体库技术获得了人源性抗角蛋白单链抗体,为临床应用研究提供了具有更广阔应用前景的人源小分子抗体。     

人源日本血吸虫Fab抗体库的构建及抗独特型抗体的筛选、鉴定

目的:构建人源日本血吸虫Fab抗体库,筛选出日本血吸虫抗独特型抗体并进行初步鉴定。方法:以3名晚期血吸虫患者切除的脾脏为源,构建人源日本血吸虫Fab抗体库。并以日本血吸虫可溶性虫卵抗原(SEA)及成虫抗原(SWAP)免疫鼠血清IgG包板进行筛选,经ELISA鉴定后,对阳性克隆进行可溶性表达。结果:构建了人源日本血吸虫Fab抗体库,库容为4.3×106,经核苷酸序列分析,证实插入片段为Fab基因片断。经过5轮筛选后,从富集的次级抗体库中随机挑取60个克隆,用ELISA法鉴定得到4个可与免疫鼠血清(Ab1)特异性结合,而不与SEA及SWAP结合的单克隆抗体(A3、B6、C4、C17),其中B6经SDS-PAGE电泳显示插入片断正确。结论:成功构建了人源日本血吸虫Fab抗体库,并从中获得人源日本血吸虫抗独特型抗体B6,为研制用于人体血吸虫病免疫预防的抗独特型抗体疫苗奠定了基础。     

基因工程技术制备和鉴定抗TNF-α蛋白Fab抗体

目的 利用基因工程技术制备人源性抗肿瘤坏死因子-α(TNF-α)蛋白的可溶性Fab抗体,并鉴定其抗原结合活性及特异性.方法 用固相化的TNF-α蛋白从人天然Fab噬菌体抗体库中筛选出表达抗TNF-α蛋白Fab抗体的阳性克隆,经酶切及连接反应构建可溶性表达噬菌粒并转化至大肠杆菌BL21(DE3)pLysS中,IPTG高效可溶性表达,SDS-PAGE及Western blot分析鉴定抗体表达情况、ELISA鉴定其抗原结合活性和特异性.结果 成功筛选并表达了人源性抗TNF-α蛋白的Fab抗体,在非还原SDS-PAGE中形成相对分子质量47 kD的条带;Western blot分析证实表达的蛋白即Fab抗体.ELISA筛选出2株抗体(TA-04,TA-13)具有良好的抗原特异性和抗原结合活性,与小牛血清白蛋白无交叉反应.结论 成功表达并鉴定了人源性抗TNF-α蛋白的可溶性Fab抗体,构建了一种基因工程抗体的有效制备途径.为基因工程抗体在肿瘤免疫治疗方面的临床应用研究奠定了基础.     

从噬菌体抗体库中筛选D二聚体特异的Fab抗体

目的:从人源性噬菌体抗体库中筛选抗人D二聚体单克隆抗体并进行可溶性表达。方法:以人D二聚体标准品为抗原对所构建的噬菌体抗体库进行两轮“吸附-洗脱-扩增”筛选,从中筛选出抗人D二聚体的噬菌体Fab抗体,并在大肠杆菌细胞中进行可溶性表达。结果:经4次电转化构建了库容为2. 8×108cfu的抗体库,滴度为4. 1×1014PFU/mL,Fab基因重组率为46%。经过淘筛后携带抗人D二聚体抗体的特异性噬菌体得到了明显的富集。用试剂盒以ELISA法检测对人D二聚体的结合活性,得到抗人D二聚体单克隆抗体。筛选出的阳性克隆成功在大肠杆菌细胞中可溶性表达筛选出的阳性克隆在大肠杆菌细胞中可溶性表达。结论:人D二聚体标准品对抗体库的淘筛,富集了表面呈现抗人D二聚体的单克隆抗体的噬菌体,成功构建了人源性抗D二聚体噬菌体抗体,并成功在大肠杆菌细胞中可溶性表达。     

大肠癌相关抗体Fab段噬菌体呈现库的表达和活性鉴定

目的利用阳性重组菌XL1-Blue-Pcomb3 表达出人源性大肠癌Fab段噬菌体原始库,固相化人大肠癌组织及细胞的相关抗原后对其进行筛选,鉴定筛选后的抗体库与人大肠癌有无特异性的结合活性。方法PCR鉴定XL1-Blue- Pcomb3重链Fd段和资链的插入率,表达出人大肠癌Fab段原始库。然后提取3例用于构建原始抗体库的致敏大肠癌组织抗原,13例非致敏大肠癌组织抗原及3种体外培养的大肠癌细胞株LoVo、HT-29、LS-174T的抗原,利用3组混合抗原分别对原始抗体库进行3轮筛选,将筛选后的3个三级库等体积混和后通过ELISA及免疫组化鉴定其与人大肠癌组织及细胞是否具有特异性的结合活性,取胃癌、食管癌及正常肠粘膜组织和胃癌、肝癌细胞进行对照研究。结果Fd片段及资链的插入率分别为40%和70%,Fd片段及资链均插入质粒Pcomb3的重组率为28%,Fab段基因库库容为2.1×106,3组大肠癌混合抗原分别对原始库进行3轮筛选后,抗体库均得到了不同程度的富集,ELISA及免疫组化显示富集后的抗体库对人大肠癌组织及体外培养的大肠癌细胞均有较特异性的结合活性。结论利用噬菌体抗体库技术筛选到了人大肠癌相关Fab段抗体群,且筛选后的抗体群与人大肠癌抗原有较特异性的结合活性。     

人源性抗血小板膜糖蛋白Ⅱb/Ⅲa Fab噬菌体抗体库的筛选与鉴定

目的从已构建的人源性抗血小板膜糖蛋白（glycoprotein,GP）Ⅱb/ⅢaFab噬菌体抗体库中筛选人源性抗血小板膜GPⅡb/ⅢaFab抗体,并对特异性阳性克隆进行鉴定。方法以表达GPⅡb/Ⅲa的CHO123细胞包板,对人源性抗血小板膜GPⅡb/ⅢaFab噬菌体抗体库进行富集筛选,采用细胞ELISA法筛选特异性阳性克隆,用Western blot法鉴定表达蛋白与GPⅡb/Ⅲa结合的特异性,将阳性克隆进行扩增、测序。结果以CHO123细胞富集淘筛3轮,并用ELISA法检测到2个高亲合力的特异性识别血小板膜GPⅡb/Ⅲa的克隆,Westernblot鉴定结果显示在阳性克隆的培养上清和细菌冻融上清均有特异性条带出现。对核苷酸序列进行同源性分析,证实这两个克隆轻链基因与人免疫球蛋白κ轻链的同源性分别高达97%和98%,重链基因与人免疫球蛋白Fd段的同源性分别高达94%和93%。结论利用噬菌体抗体库技术,成功地从人源性抗血小板膜GPⅡb/ⅢaFab抗体库中筛选出抗血小板膜GPⅡb/Ⅲa的特异性阳性克隆,该克隆具有较高的特异性,其克隆基因符合抗体可变区结构特点。     

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.     

A preliminary screening study on the associated proteins in human psoriasis vulgaris by serum proteomics technologies

Objective：To investigate the optimum screening conditions of associated proteins in human psoriasis vulgaris by serum proteomics technique, and to screen the different expression proteins related with psoriasis vulgaris. Methods：Serum samples of peripheral blood were collected from newly diagnosed psoriasis vulgaris patients in the clinic, and 20 matched healthy persons. Serum albumin IgG was removed by filtering with ProteoExtract Albumin/IgG. After comparative proteomics analysis the different protein spots were identified using 2-DE and MS. Results ：Electrophoresis figures with high resolution and reproducibility were obtained. Three different expression proteins were found only in the serum from psoriasis vulgaris patients, while nine other different proteins expressing from healthy volunteers. Conclusion：The protein expression was different in the serum between the psoriasis vulgaris patients and healthy volunteers. It was hoped that we could find the biomarkers related to psoriasis vulgaris by using proteomics.     

A CASE OF HUGE GASTRIC DISOPYROBEZOAR

PYTOBEZOARS are common bezoars in gastrontestinal tract, commonly seen in stomach and mall intestine,1but huge disopyrobezoars are     

Acupuncture Treatment of Chest Bi Syndrome

CASE HISTORY A male,a retired officer of 61 years old,paid his first visit on May 12,2006.The patient stated that 2 weeks before he suddenly got paroxysmal precordial choking pain,with irregular attacks.Each attack would last several seconds to several minutes.The attack was accompanied with heavy oppressed sensation in the precordial region,palpitation,lassitude,and in severe cases,sweating,which was once diagnosed by a hospital as'coronary heart disease'.Recalling the case history,the patient said that several days before he got angry with others because of some affairs in work,and he was not so used to the life pattern since he had just retired from his busy working post.At usual times,he was quick tempered and peevish.In recent days,he had poor appetite,and loose stool.     

Instructions to Contributors

The English edition of Chinese Journal of Integrative Medicine is edited and published by the Chinese Journal of Integrated Traditional and Western Medicine Press.Original scientific papers of advanced clinical and experimental medicine in the field of complementary/alternative medicine or integrative medicine are expected.     

Development Is A High Level Inheritance for China Time-honored Brand Products

<正>As culture is the soul of a nation,the three books,"History of Chinese Philosophy,""History of Chinese Culture"and"History of Chinese Science and Technology"have demonstrated the superiority of our great Chinese culture,and its significant impact in the field of international culture.Both core value and practical value of the Chinese culture are very clear,reflected in the views of universe,nature,life,health,disease,and therapy and related considerations,concepts,and technical methods.