乳鼠成骨细胞酵母双杂交cDNA文库构建

目的构建乳鼠成骨细胞的酵母双杂交cDNA文库。方法用TRIzol法提取乳鼠成骨细胞总RNA,按照SMART cDNA Library Construction Kit ( CLONTECH)说明书反转录合成双链cDNA.Spin Column去除短片段,然后用同源重组的方法将双链 cDNA和PGADT7~rec载体共转化到酵母细胞Y187中,建立文库并计算文库滴度。结果提取的总RNA的A 260/A 280为 1.99,琼脂糖凝胶电泳示28SrRNA、18SrRNA2条带,且28S与18S带亮度比值约为2。酵母文库库容为1. 68 x 107,重组率为 100%。插人片段PCR检测提示大小分布为1. 5 -4. 0 kb,平均长度约为3. 0 kb。结论乳鼠成骨细胞酵母双杂交cDNA文 库构建成功。     

肿瘤坏死因子α转化酶胞内区诱饵质粒的构建及鉴定

目的:构建用于酵母双杂交系统的诱饵质粒载体pGBKT7-TACEc(肿瘤坏死因子α转化酶胞内区),并检测其是否具有自身激活及毒性作用。方法:从BALB/c雄性小鼠睾丸组织提取总RNA,用RT-PCR方法获得TA-CEc,克隆于pGBKT7载体上,酶切鉴定及序列分析,并检测pGBKT7-TACEc在酵母双杂交系统中的自激活和毒性作用。结果:实验成功构建了诱饵质粒载体pGBKT7-TACEc,并证明其在酵母双杂交系统中无自激活及毒性作用。结论:pGBKT7-TACEc可应用在酵母双杂交系统中,寻找小鼠睾丸cDNA文库中与TACEc相互作用的蛋白质,为筛选小鼠睾丸中与TACEc相互作用的蛋白奠定了重要基础。     

减数分裂重组关键蛋白MLH1结合蛋白的酵母双杂交筛选和鉴定

目的探寻减数分裂关键重组蛋白MLH1在精子发生中的结合蛋白。方法以MLH1为诱饵,通过酵母双杂交系统,在小鼠睾丸组织的cDNA文库中进行筛选,寻找MLH1的相互作用蛋白并通过GST pull-down和免疫共沉淀的方法进行验证。结果通过酵母双杂交的筛选,在小鼠睾丸中经四缺板筛出蛋白29个,包括己知的MLH3和PSM2等。通过GST Pull-down和免疫共沉淀验证了其中一个新的蛋白RHNO1与MLH1的结合。通过Real-time PCR检测发现RHNO1在睾丸中高表达,提示RHNO1在精子发生中具有重要功能。结论酵母双杂交系统是寻找结合蛋白的有力手段,发现了新的MLH1结合蛋白RHNO1,可能参与了减数分裂同源重组。     

国人正常睾丸组织定向克隆表达cDNA文库的构建及意义

目的　研究精子发生相关基因的结构与功能 ,构建中国人正常睾丸组织定向克隆表达cDNA文库。　方法　提取国人正常睾丸组织mRNA ,逆转录合成cDNA ,SalI/NotI酶切后基因重组法定向克隆于质粒表达载体pSPORT1中 ,转化细菌后扩增。　结果　文库大小为 7× 10 5,SalI/NotI酶切鉴定 ,插入的cDNA片段多为 0 .5～ 2 .0kb。　结论　国人正常睾丸组织定向克隆表达cDNA文库构建成功 ,质量较好 ,可进行包括低丰度mRNA在内的所有丰度mRNA的cDNA克隆筛选 ,为进一步研究精子发生相关基因的结构与功能提供了实验依据。     

肾癌酵母双杂交文库的构建及其意义

目的　研究肾癌中的信号传导尤其是Wnt/Frizzled信号通路在肾癌发生、发展中的作用 ,寻找具有生物意义的小肽 ,构建肾癌的酵母双杂交文库。　方法　提取肾癌组织的mRNA ,逆转录合成cDNA ,SalI/NotI酶切后基因重组法定向克隆于穿梭质粒pPC86中 ,转化DH5α菌后扩增。　结果　文库大小为 6× 10 6,SalI/NotI酶切鉴定 ,插入cDNA片段多为 1～ 3kb。　结论　肾癌pPC86酵母双杂交文库的构建成功为研究肾癌发生机理、诊断和治疗 ,尤其是信号分子通路治疗提供了有效的工具。     

SMART技术构建恒河猴睾丸组织全长cDNA文库

目的 运用SMART技术构建恒河猴睾丸组织全长cDNA文库.方法 提取恒河猴睾丸组织总RNA并分离出 mRNA,用clontech公司SMARTMcDNA文库构建试剂盒反转录合成第一链cDNA,LD-PCR扩增获得全长cDNA双链;经SfiI酶切、层析柱分离后,500bp以上的片段与pDNR-LIB连接后电转化及铺板,建成原始文库.结果 经鉴定,原始文库为2.5×106个重组子,扩增后文库滴度为4.5 X 109CFU/mL.重组率为100%,平均插入片段为1.6kb.结论 已构建文库质量较高,为进一步筛选、克隆睾丸特异表达基因奠定了基础.     

人睾丸cDNA文库的构建

本研究从人睾丸组织的总RNA中纯化mRNA,并以此为模板,在AMV反转录酶的作用下,制备互补的SS-cDNA。用RNaseH降解模板mRNA,经DNA多聚酶Ⅰ作用,合成ds-cDNA。用T_4DNA多聚酶制备cDNA的平末端,在T_4DNA连接酶的作用下,连接Ecorl接头并与λgt11载体重组连接,以大肠杆菌Y1090做受体菌转染,构建人睾丸cDNA文库。结果表明cDNA文库构建成功,将对生殖和避孕进一步研究发挥重要的作用。     

肾癌组织消减文库的构建与肾癌特异表达基因克隆

目的 构建人肾癌组织与正常肾组织差异表达的cDNA消减文库,从文中克隆鉴定出肾癌特异性表达的基因奠定基础。方法 应用抑制性消减杂交技术,分别从肾癌及正常肾组织中提取poly(A) RNA;依次合成单链及双链cDNA,分别与2种不同的接头衔接,再与正常肾cDNA进行2次消减杂交及2次抑制性PCR;将产物T／A载体连接接构建成功cDNA消减文库。结果 构成功具有高消减效率的人肾癌组织cDNA消减文库,文库扩增后得到350个阳性克隆,其中95％克隆均含50－400bp插入片段。结论 应用抑制性消减杂交技术所构建的人肾癌组织cDNA消减文库为进一步大批量筛选、克隆肾癌特异性表达的基因奠定了基础。     

HBV表面抗原大蛋白与C53蛋白在肝细胞内的相互作用研究

目的 采用酵母双杂交方法筛选出肝细胞内HBV表面抗原大蛋白(LHBs)的结合蛋白,并验证LHBs与C53蛋白在肝细胞内的相互作用.方法 构建pGBKT7-LHBs作为诱饵质粒,从人肝脏cDNA文库中筛选与其相互作用蛋白的编码基因,发现其中包括C53基因.分别构建pCMV-5a-C53和pACT-C53质粒,采用哺乳动物双杂交及免疫共沉淀的实验方法验证肝癌细胞系HepG2内这两种蛋白之间的相互作用.结果 成功从肝脏cDNA文库筛选出C53蛋白;成功构建了pCMV-5a-C53和pACT-C53质粒,并通过哺乳动物双杂交及免疫共沉淀方法明确了LHBs及C53在肝细胞内的相互作用.结论 肝癌细胞系HepG2中LHBs及C53存在明确的相互作用,为进一步研究二者的相互作用及对相关生物学功能的影响奠定了前期基础.     

应用酵母双杂交系统筛选小鼠PIAS2相互作用蛋白

目的:应用酵母双杂交技术,从小鼠精原细胞cDNA文库中筛选与PIAS2(protein inhibitor of activatedSTAT2,PIAS2)相互作用的未知蛋白。通过对其相互作用蛋白的筛选和研究,探讨PIAS2在精子发生中的作用机制,为临床男性不育症的治疗和诊断提供理论基础。方法:以pGBKT7-PIAS2为诱饵质粒,从小鼠精原细胞cDNA文库中筛选出与pGBKT7-PIAS2相互作用的阳性克隆,对验证后的阳性克隆进行测序及生物信息学分析,进一步采用直接酵母双杂交进行相互作用的验证。结果:经过筛选、测序、同源性分析及直接酵母双杂交验证,发现了与PIAS2相互作用的8个候选蛋白:Cyfip2、Psmb3、Nme1、nischarin、Nsun5、Ints10、Gnb2l1及Ndufaf3。结论:应用酵母双杂交系统,共筛选得到了8个不同的基因,其编码蛋白与PIAS2相互作用,可能与男性生殖调控相关。对上述蛋白与PIAS2的相互作用研究有助于揭示PIAS2的作用机制。     

人Lipocalin型前列腺素D合成酶真核载体的构建及酵母表达菌株的建立

目的 :在毕赤酵母中表达人睾丸Lipocalin型前列腺素D合成酶 (L PGDS) ,以利于生物学功能及临床应用研究。　方法 :用PCR的方法从质粒pGEX 2T htL PGDS上扩增出人睾丸L PGDS基因编码序列 ,构建该基因的酵母表达质粒 ;将表达质粒电转化毕赤酵母 ,甲醇诱导L PGDS的表达。　结果 :PCR扩增的L PGDS成熟肽基因编码序列克隆T载体后 ,经测序证明与所报道的人睾丸L PGDScDNA完全一致。对培养上清进行SDS PAGE分析显示 ,在相对分子质量为 2 70 0 0处有重组蛋白的表达 ,与理论值完全一致。　结论 :人Lipocalin型前列腺素D合成酶在毕赤酵母中获得了高效、分泌性表达     

利用重构pc-MDR1质粒快速诱导肝癌细胞耐药

目的：从裸鼠原位耐药肿瘤组织中进行MDR1 cDNA的全克隆和pc-MDR1重组质粒的构建，并转染HepG2细胞，快速诱导其耐药，并筛选其抗性克隆。方法：设计单酶切位点引物，利甩长距离逆转录PcR(Long RT-PCR)技术，由HepG2耐药细胞扩增MDR1 cDNA，约3．8kb大小。将其插入至真核表达载体pcDNA3．0质粒中构建pc-MDR1诱导质粒，使其能够在真核细胞中表达p-gp蛋白。转染HepG2细胞，并用G418筛选出转染的细胞。结果：成功地扩增出3．8kb左右的MDR1 cDNA片段，经酶切鉴定、RT-PCR扩增特异片段和序列测序结果显示质粒构建初步成功，用G418筛选细胞耐药抗性克隆的时间约为14d。结论：从耐药肿瘤组织中进行MDR1 cDNA的全克隆和pc—MDR1诱导质粒的构建成功快速诱导了肝癌细胞发生耐药，为进一步研究肿瘤细胞的耐药机制奠定了很好的基础。     

Construction and characterization of a cDNA library from liver tissue of Chinese Banna minipig inbred line

A xenograft that performs efficient functions is an essential premise for successful xenotransplantation. Our early study indicated that Chinese Banna minipig inbred line (BMI) was an ideal xenograft donor. However, the activities of some proteins synthesized by the BMI liver are different from the human, which could lead to functional disorders in coagulation, fibrinolysis, and anticoagulation after liver xenotransplantation. Therefore, it is important to investigate the genetic background of protein incompatibility and to provide new strategies for gene manipulation. In this study we constructed a cDNA expression library using BMI liver tissue to obtain an understanding of nucleic acid and protein differences between the two species. We extracted total RNA and purified mRNA of the liver tissue from one of the sixteenth inbred generation of BMI/JS 151 substrain. After double-strand cDNA synthesis, we fractionated it on a CHROMA APIN-400 column; ligated the longer than 500bp cDNA into a ZAP Express Vector; and performed a lambda: phage packaging reaction, library amplification, and titer. We randomly picked 12 plaques and tested the length of inserts. The titers of the primary and amplified libraries were 1.0 x 10(6) pfu/mL and 5.0 x 10(9) pfu/mL, respectively. The percentages of recombinants were 97.0% in the primary library and 98.0% in the amplified library. The lengths of most inserts were between 750 bp and 2.0 kb. Thus, we successfully constructed a cDNA expression library from BMI liver tissue. Using the library, we hope to get a full-length cDNA of some important genes and conduct further studies on porcine liver function in xenotransplantation.     

Identification of a novel testis-specific gene and its potential roles in testis development／spermatogenesis

Aim: To identify and characterize a novel gene with potential roles in testis development and spermatogenesis.Methods: A cDNA microarray was constructed from a human testis large insert cDNA library and hybridized with probes of human or mouse adult and fetal testes. Differentially expressed genes were isolated and sequenced. RT-PCR was used to test the tissue distribution of the genes of interest and in situ hybridization was performed to localize the gene expression in the mouse testis. A range of bioinformatical programs including Gene Runner, SMART, NCBI Blast and Emboss CpGPlot were used to characterize the new gene‘s feature. Results: A novel testis-specific gene,NYD-SPS, was differentially expressed in fetal and adult testes. The deduced protein structure of NYD-SP5 was found to contain an IQ motif (a short calmodulin-binding motif containing conserved lie and Gin residues), a Carbamate kinase-like domain, a Zn-dependent exopeptidase domain and a lactate dehydrogenase (LDH) C-terminal-like domain. RT-PCR analysis revealed that NYD-SP5 was predominantly expressed in the testis but not in other 15 tissues examined. In situ hybridization and RT-PCR examinations revealed that the expression of NYD-SP5 was confined in the male germ cell but not present in the somatic cell in the testes. Conclusion: NYD-SP5 is a newly found testisspecific gene with potential roles in testis development and spermatogenesis through a calmodulin-activated enzyme.     

人骨形成蛋白7基因重组35型腺病毒载体的构建与鉴定

目的探讨构建携带人骨形成蛋白7(human bone morphogenetic protein-7,hBMP-7)基因的重组35型腺病毒(serotype35adenovirus,Ad5F35)载体,为基因治疗研究提供一种新的方法,并观察其在兔骨髓间充质干细胞(MSCs)中的表达。方法以pcDNA1.1/Amp-hBMP7质粒为模板扩增骨形成蛋白(BMP-7)基因(1.3kb),将回收的PCR产物片段克隆入pDC316载体,获得重组质粒pDC316-hBMP7。骨架质粒pBHG-fiber5/35和穿梭质粒pDC316-hBMP7共转染293细胞,同源重组产生重组腺病毒rAd5/F35-hBMP7。同样的方法构建rAd5/F35-EGFP。在体外用不同感染复数(M.O.I)值的rAd5/F35-hBMP7和rAd5/F35-GFP分别转染兔MSCs,并留置空白对照。采用流式细胞仪、RT-PCR方法检测目的基因的转染效率和表达。结果PCR、酶切鉴定以及序列测定与比对分析表明,重组腺病毒rAd5/F35-hBMP7质粒构建正确。转染兔MSCs的最高效率可达99%,hBMP-7基因存在于转染后的MSCs中并表达相应的mRNA。结论本实验成功构建了含hBMP-7基因的重组腺病毒rAd5/F35-hBMP7载体,在体外能高效转染兔MSCs。     

人骨形成蛋白7基因重组2型腺相关病毒载体的构建及其在骨髓间充质干细胞中的表达

目的构建人骨形成蛋白7基因（hBMP7）重组腺相关病毒载体,并观察其在兔骨髓间充质干细胞中的表达。方法将骨形成蛋白7基因片段克隆入穿梭质粒pUC18获得重组质粒pUC18-hBMP7。KpnⅠ和鼠Ⅱ双酶切质粒pUC18-hBMP7／与pSNAV,用T4DNA连接酶连接分别回收的两片段后转化大肠杆菌DH5α感受态细胞,获得重组质粒PSNAV—hBMP7／,转染BHK-21细胞,筛选培养,用能表达Rap和Cap的重组Ⅰ型单纯疱疹病毒HSVl-rc／△UL2感染此细胞,裂解细胞收获病毒液。采用氯仿处理-PEG／NaCl沉淀-氯仿抽提法分离浓缩、纯化与测定病毒滴度。用rAAV2-hBMP7／和rAAV2-EGFP在体外分别转染兔骨髓间充质干细胞。流式细胞仪、RT-PCR和Western—blot方法检测兔骨髓间充质干细胞中hBMP-7基因的转录和表达。结果成功构建具有感染活性的重组腺相关病毒载体rAAV2-hBMP7／,病毒载体对兔骨髓间充质干细胞早期转染效率可达99．8％,hBMP7／在兔骨髓间充质干细胞中可得到转录和表达。结论成功构建了人骨形成蛋白7基因重组腺相关病毒载体,rAAV2-hBMP7／载体在体外可转染兔骨髓间充质干细胞,并获得较高的转染效率。     

A strategy for identifying candidate sperm antigens for immunocontraception: isolation of human testis cDNA clones using polyclonal antisera directed against hamster acrosomal membrane preparation

Components of the mammalian sperm acrosome that have been conserved during evolution are probably essential for fertilization and are therefore potential antigens for the development of an immunocontraceptive vaccine. In order to identify such protein components, a series of specific polyclonal antisera were generated by immunizing rabbits with purified acrosomal membrane fractions from hamster epididymal spermatozoa. Antisera were finally selected using immunological and in-vitro fertilization assays, and used to then screen a human testis Λgt11 cDNA library. As a result of this screening over 70 clones were identified, selected and purified. The cDNAs were amplified by polymerase chain reaction (PCR) and the inserts characterized by restriction enzyme digestion and oligonucleotide probing techniques. The functional activity of β-galactosidase fusion proteins expressed by these clones was tested by a hamster in-vitro fertilization assay (IVF). Fusion protein from three clones (HA5-2, HA6-2 and HB4-1) inhibited significantly fertilization and reduced spermzona binding compared to controls. To date, sequence data has been obtained from HB4-1 (1.75 kb). The first 1132 nucleotides displayed > 96% homology to human testis-specific lactate dehydrogenase (LDH-C4) gene, the product of which is a known candidate antigen for a contraceptive vaccine. This finding suggests that a strategy involving the screening across species for conserved moieties of the mammalian acrosome may be useful for identifying candidate antigens for immunocontraception.