人神经营养素-3成熟蛋白基因克隆及序列分析

应用 PCR技术 ,分别以 2个健康成人末梢血白细胞染色体 DNA为模板 ,扩增出人神经营养素 - 3( h NT- 3)成熟蛋白基因 ,并克隆至原核质粒 p UC1 9中进行基因序列测定。将所得序列与 Gen Bank提供的序列 ( M6 1 1 80 )进行对比 ,结果显示一序列与已知序列完全一致 ,另一序列中第 1 41、2 0 4和 2 1 9位碱基发生了改变 ,但改变的碱基不影响其编码的氨基酸。     

MOLECULAR CLONING OF HUMAN NEUROTROPHIN-4 GENE

Ｎｅｕｒｏｔｒｏｐｈｉｎ 4 (ＮＴ 4 )ｉｓａｍｅｍｂｅｒｏｆｈｅｎｅｕ ｒｏｔｒｏｐｈｉｎｆａｍｉｌｙｏｆｓｔｒｕｃｔｕｒａｌｌｙｒｅｌａｔｅｄ ｐｒｏｔｅｉｎｓ[1] ,ｔｈａｔａｌｓｏｉｎｃｌｕｄｅｓｎｅｕｒｏｔｒｏｐｈｉｎ 3(ＮＴ 3) ,ｎｅｕ ｒｏｔｒｏｐｈｉｎ 6 (ＮＴ 6 ) ,ｎｅｒｖｅｇｒｏｗｔｈｆａｃｔｏｒ (ＮＧＦ)ａｎｄｂｒａｉｎ ｄｅｉｖｅｄｎｅｕｒｏｔｒｏｐｈｉｃｆａｃｔｏｒ (ＢＤＮＦ) [2 - 6] ,ｗｈｉｃｈｓｕｐｐｏｒｔｓｔｈｅｓｕｒｖｉｖａｌｏｆｖｅｒｔｅｂｒａｔｅｎｅｕｒｏｎｓ[7] .Ａｔｐｒｅ…     

CLONING AND SEQUENCING OF MATURED FRAGMENT OF HUMAN NEVER GROWTH FACTOR GENE

Humannervegrowthfactor (hNGF)istheear liestrevealedcellulargrowthregulator.Researchershaveattainedafairlygoodunderstandingofthisnu tritionalfactorthatisessentialtothesurvival,growthanddifferentiationofcentralandperipheralnerves.Theclinicaluseofnerve growthfactor(NGF)hasapotentialtorepairorregrowdamagednerves,topreventandtreatretrogradeneurologicaldiseases,andto promotedifferentiationofneuro blast .Thepreviousstudieshaveshownthatdirectpu rificationofNGFisdifficultandoflessproductionwhilegen…     

THE DETECTION OF P53 GENE IN HUMAN CERVICAL CARCINOMA WITH AND WITHOUT HUMAN PAPILLOMAVIRUS INFECTION

ＴＨＥＤＥＴＥＣＴＩＯＮＯＦＰ５３ＧＥＮＥＩＮＨＵＭＡＮＣＥＲＶＩＣＡＬＣＡＲＣＩＮＯＭＡＷＩＴＨＡＮＤＷＩＴＨＯＵＴＨＵＭＡＮＰＡＰＩＬＬＯＭＡＶＩＲＵＳＩＮＦＥＣＴＩＯＮＳｕｎＹｉ（孙毅）；ＳｉＬｕｓｈｅｎｇ（司履生）ＳｕｎＹｉ；ＳｉＬｕｓｈｅｎ...     

应用PCR技术克隆人乳头瘤病毒16型E6基因

为进一步研究与宫颈癌发生密切相关的人乳头瘤病毒16型E6基因的转化作用,我们运用PCR技术扩增HPV16 E6 DNA,以pUC—19为载体,大肠杆菌了JM103为宿主菌,组建了质粒pRCE6经限制性核酸内切酶和Southern转印杂交证实,其插入片段约为0.5Kb,含有全部HPV16 E6序列。该质粒的组建为检测HPV感染中mRNA转录提供了特异性的早期基因探针。同时为进一步研究HPV16 E6基因的转化作用及转化蛋白打下基础。     

DETECTION OF BCL-2 GENE MAJOR BREAKPOINTREGION REARRANGEMENT IN HUMAN B-CELL LYMPHOMAS

Thet(l4;18)chromosomaltranslocationsarerecognizedasacytogeneticabnormalityinB-celllymphomas,especiallyinfollicularlymphomas',>.Aputativeoncogeneonchromosome18bandq2ltermedB-cellleukemia-lymphoma-2(bcl-2)isjux-taposedtosegmentsoftheimmunog1obulin(Ig)heavy-chaingenelocatedonchromosome14bandq32.Bcl-2rearrangementsarecommonlyfoundinfollicularlymphomasintheAmericant2i.Howev-er,theconflictingresultsregardtoincidenceofthetranslocationinJapaneseB-ce1llymphomashasyieldedusingcytogeneticana1ysisandSo…     

CLONING OF HUMAN PAPILLOMAVIRUS 16 GENOME FROM CERVICAL CANCER TISSUE

Humanpapillomavirus(HPV)caninducemanybenignproliferateddiseases,forexample,lowergenitalwartsandhasacloserelationshipwithcervicalcancer.Morethan70typesofHPVhavebeenclonedandidentifiedc1J.MostofthemwereclonedintoplasmidPBR322.SinceHPVcannotproliferateinanycellcultureinvitroasyetmostHPVDNAsusedinChinacomefromabroad.InordertoconstructourHPVDNAclone,theHPV-16DNAwasisolatedfromthecervicalcancertissueandclonedasfollows.MATERIALSANDMETHODS1SpecimencollectionandtissueDNAextraction…     

胎儿SRY基因用于产前诊断的初步研究

采用ＰＣＲ技术,对５０例胎儿组织ＤＮＡ进行了Ｙ染色体特异的ＳＲＹ基因扩增。扩增片段长度为２５０ｂｐ。结果显示：３２例早孕绒毛ＤＮＡ中,有１７例扩增出特异性片段．１５例未扩增出特异性片段,有、无特异性片段比例为１．１３３：１,接近胎儿自然出生性别之比;１８例中孕引产胎盘ＤＮＡ中８例阳性,１０例阴性,与引产胎儿实际性别完全一致。因ＰＣＲ扩增胎儿ＳＲＹ基因特异性强、灵敏度高,可用于早期胎儿性别的产前诊断。     

STUDY OF ECK GENE EXON-3 FROM HUMAN NORMAL TISSUE AND BREAST CANCER CELL LINE

ECK (epithelialcellkinase) ,alsonamedasEphA 2 ,isoneofthemembersofEphAsubfamilyinEphfamily .ECK protein ,atransmembranety rosinekinase[1] ,canbestructurallydividedintothreedomainsaccordingtotheirpositionsincells.ThreedomainsareexternaldomainwithanN ter minalsignalpeptide ,atransmembranedomainandacytoplasmicdomainwhichincludesacanonicalpro tein tyrosinekinasecatalyticdomain .ECKiswide lyexpressedinavarietyofhumantissues ,abun dantly presentinlung ,skin ,smallintestineandovary .ECKisalsoe…     

RAPID MOLECULAR DETECTION OF METHICILLIN-RESISTANT S. AUREUS IN THE INTENSIVE CARE UNIT

Staphylococcus aureus is found in the nose andon the skin of a variety of healthy individuals,andis an opportunistpathogen in patients with loweredhost resistance.The isoxazolylpenicillin (e.g.me-thicillin,cloxacillin,flucloxacillin) have been themainstay of treatment for infection over2 5years.But emergence of antibiotic resistance(MRSA) is aproblem in hospitals due to transmission of epi-demic MRSA strains with antibiotic resistance.Ac-quisition of MRSA continues to be of major con-cern…     

CLONING AND EXPRESSION OF A cDNA SEQUENCE FOR HUMAN THIOREDOXIN

Thioredoxin (TRX)isanoxidoreductaseen zyme,withamolecularweightof 1 2kD .Itwasi dentifiedoriginallyinE .coliasanhydrogendonorforribonucleotidereductaseanddeoxyribonucle otidesynthesis[1] .Thegeneofhumanthioredoxin(hTRX)islocatedonchromosome 3 p1 1 p1 2 ,withafulllengthof 1 3Kb .Theopenreadingframe ( 3 1 5nucleotideslong)codedforaproteinof 1 0 4aminoacids[2 ,3] .ManyfunctionsofTRXhavebeenre ported,whichincludethefollowing :TheTRXsystem (whichincludesHADPHasaprotondonor,TRXreductase ,…     

CLONING AND SEQUENCING OF MATURE FRAGMENT OF HUMAN BMP4 GENE

Bone morphogenetic protein- 4(BMP- 4) is alow molecular weight glycoprotein ,classified as amorphogen.It is capable of inducing the formationof new cartilage and bone.This osteoinductiveability has led to the use of bone morphogeneticproteins as therapeutic agents for creation of newbone useful in treatment of skeletal injuries anddiseases,and in oral and maxillofacial applica-tions.Although many researchers have got the na-tive BMP from animal s demineralized bone by bio-chemical method,the…     

宫颈癌组织表皮生长因子受体对TKI敏感性相关的基因突变研究

目的研究宫颈癌组织表皮生长因子受体(epidermal growth factor receptor,EGFR)基因中是否存在与EGFR酪氨酸激酶抑制剂(tyrosine kinase inhibitor,TKI)药物敏感性相关的18、19及21外显子突变,为本地区宫颈癌的靶向治疗提供依据。方法收集70例宫颈癌患者的新鲜癌组织标本,提取DNA,以特异性引物PCR扩增EGFR基因外显子18、19及21,进行DNA测序并分析序列相似性。结果 70例宫颈癌组织提取质量良好的DNA中,均未检测到EGFR基因18、19及21外显子突变。结论宫颈癌组织EGFR基因外显子18、19及21突变罕见。     

人乳头瘤病毒11型E6基因完整开放阅读框架的克隆和鉴定

目的　为构建人乳头瘤病毒 1 1型 (HPV1 1 )DNA疫苗 ,从尖锐湿疣病变组织中克隆疫苗靶基因E6。方法　利用改良提取染色体外游离DNA的方法制备模板DNA ,采用聚合酶链反应(PCR)技术进行基因克隆。结果　从病变组织中扩增出了全长人乳头瘤病毒 1 1型E6基因完整开放阅读框架 ,并插入中介载体T easy中构建重组质粒 ,转化JM1 0 9扩增后经酶切、PCR及测序分析 ,证实克隆成功。结论　该实验的成功为下一步制备HPV1 1的治疗性疫苗奠定基础。     

人脑源性神经营养因子基因克隆及序列分析

目的　克隆人脑源性神经营养因子 (hBDNF)基因并进行序列分析。方法　提取健康成人末梢血白细胞基因组DNA作为模板 ,应用PCR技术和T 载体克隆法克隆hBDNF基因 ,筛选阳性克隆、酶切鉴定 ,并进行序列测定和分析。结果　DNA序列测定的结果与GenBank提供的已知序列 (M6 1 1 81 )比较 ,所克隆的hBDNF基因从起始密码子ATG到终止密码子TAG全长共744bp ,序列完全相同。结论　自人基因组DNA中克隆hBDNF基因 ,为进一步开展阿尔茨海默病 (AD)的基因治疗积累了资料。     

涎腺肿瘤组织中人乳头瘤病毒DNA的检测

为探讨腺上皮起源的涎腺良、恶性肿瘤与人乳头瘤病毒（Ｈｕｍ ａｎ ｐａｐｉｌｌｏ ｍ ａｖｉｒｕｓ, ＨＰＶ）感染的关系,应用聚合酶链反应技术（ＰＣＲ）,采用共有引物检测４４例涎腺肿瘤组织中的ＨＰＶ ＤＮＡ的存在状况。１０ 例正常涎腺组织作为对照。结果显示：肿瘤组织中ＨＰＶ ＤＮＡ 阳性检出率达７０．４５％ （３１／４４）,其中恶性肿瘤 ＨＰＶＤＮＡ 阳性率为７７２７％ （３１／４４）;良性肿瘤ＨＰＶＤＮＡ阳性率为６３６３％ （１４／２２）;而正常对照组织中ＨＰＶ ＤＮＡ阳性率仅为１０％ （１／１０）。提示：涎腺肿瘤的发生可能与ＨＰＶ感染有关     

人B7.2 cDNA的克隆及其真核表达

为克隆人 B7.2 c DNA,构建 B7.2真核表达质粒 ,并在哺乳动物细胞中表达。分离正常人淋巴结淋巴细胞 ,经 LPS诱导后 ,以 RT- PCR,克隆 B7.2 c DNA;构建真核表达质粒 p BCMGSNeo- B7.2 ,转染哺乳细胞 ,进行表达。结果成功克隆了 B7.2c DNA,并经测序证实 :所构建的 B7.2抗原真核表达质粒可在哺乳动物细胞中高效表达。表明经 LPS诱导的人淋巴细胞可表达 B7.2 m RNA,所建立的 p BCMGSNeo-B7.2哺乳动物真核表达质粒 ,可供进一步研究 B7.2结构和功能。     

THE CONSTRUCTION AND EXPRESSION OF THE MURINE SCFV GENE IN E. COLI AGAINST HUMAN CERVICAL CANCER

Objective To obtain the gene of murine Single chain Fv fragment （ScFv） against haman cervical cancer and to express it in E. coli. Methods The variable region gene fragments of the heavy and light chains, which were amplified respectively using recombinant DNA techniques from CsA125 hybridama cells, were spliced together through a flexible linker to ScFv against human cervical cancer. The ScFv genes were then cloned into expression vector pCANTAB 5E and expressed in E. coli HB2151 and TG1 respectively. The soluble ScFv were characterized by SDSPAGE and Western blot. The antigen-binding activities of the soluble and phage displayed ScFv were assayed by ELISA and cell immunohistochemical analysis. Results The expressed ScFv antibodies were soluble and phage displayed. soluble ScFv secreted and expressed in E. coli HB2151 induced by IPTG were confirmed with SDS-PAGE, Western blot and ELISA. The specific binding capacity of the soluble and phage displayed ScFv to the surface associated antigen of human cervical cancer cell line was further confirmed with immunohistochemical studies. Conclusion The soluble and phage displayed ScFv expressed in E. coll against haman cervical cancer showed high, specific affinity for the cervical cancer cell line surface associated antigen.     

结核患者不同标本中结核杆菌DNA的检测

为提高不典型结核患者的诊断符合率，寻求结核病理想的实验室诊断技术，运用聚合酶链反应技术对60例结核病患者7种（125份）不同标本中的结核杆菌DNA进行了检测。结果显示疾液、胸水中的DNA阳性率较高（分别为54％、49．25％），血清中的DNA阳性率仅为28％（P＜0．05），其原因主要与不同标本中存在的结核杆菌数目的多少有关。同一标本被检测两次（复检），阳性例数较前有所提高，这样可使某些假阴性结果得到及时纠正。如果同一患者多种标本检测结果同时为阳性，则更支持结核病的诊断。     

THE EUKARYOTIC EXPRESSION OF HUMAN PERFORIN PROTEIN AND THE ESTABLISHMENT OF HYBRIDOMAS PRODUCING ANTI-HUMAN PERFORIN PROTEIN

Perforin is expressed mainly in activated cyto-toxic T lymphocytes (CTLs ) and natural killer(NK) cells. The human perforin gene has beencloned. The full length of its cDNA is l668bp, andthe mature HP protein is made up of 534 aminoacid residues with a molecular weight of 66Kd~75Kd. In CTLs and NK cells, perforin is stored incytoplasmic granules and is a major effector of cy-tolysis by these cells. Upon granules releasing,perforin monomers insert into the plasma mem-branes of target ce…