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。     

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。     

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。     

从人肾癌酵母双杂交文库中筛选TCF4相关蛋白

目的：应用酵母双杂交技术寻找在肾癌发生过程中与Wnt信号家族成员T细胞因子4(TCF4)相互作用的新蛋白。方法：以Wnt通路中的关键信号分子TCF4的N端和DNA结合域(TCF4ND，1～495aa)为诱饵蛋白，筛选肾癌双杂交文库，得到的阳性克隆酶切分类，测序后在Gen-Bank中进行Blast分析。结果：筛出与hTCF4相互作用的阳性克隆51个，包括β环连蛋白(β-catenin)，hTCF4，激活转录因子5(ATE5)，人类锌指蛋白等，其中未知基因2个。结论肾癌的发生、发展可能有多种信号通路参与调控。     

丁酸钠诱导MCF-7细胞凋亡反义cDNA文库的构建和初步鉴定

目的构建丁酸钠诱导McF-7细胞凋亡过程的反义cDNA文库,以分离丁酸钠抗肿瘤效应基因。方法收集经2．5mmol/L丁酸钠处理0、12、18、24、36、48、72h的人乳腺癌MCF-7细胞,提取poly(A)+RNA,逆转录合成cDNA第一链和第二链,双链cDNA修平末端后连上EcoRⅠ/HindⅢ接头,分别用EcoRⅠ/HindⅢ酶切消化后,反向连上载体pcDNA 3．1,连接产物转化大肠杆菌得一反义cDNA文库,随机挑取40个克隆子进行酶切鉴定。结果构建的反义cDNA文库含1×10~6重组子,重组效率为90%,插入子平均长度为1．5kb。结论构建的文库容量较大,质量较高,为进一步分离丁酸钠抗肿瘤效应基因奠定了基础。     

人前列腺癌cDNA文库的构建

目的采用高效互补ＤＮＡ（ｃＤＮＡ）合成技术构建人前列腺癌的ｃＤＮＡ文库。方法从前列腺癌患者的经尿道电切标本中提取总ＲＮＡ，纯化后得到Ｐｏｌｙ（Ａ）＋ＲＮＡ；依次合成单链及双链ｃＤＮＡ；经纯化后与衔接头相连接；再与噬菌体载体λｇｔ１０臂相连接，于体外包装后建成ｃＤＮＡ文库；以大肠杆菌Ｃ６００ｈｆｌ－行滴度测试及扩增。结果构建的人前列腺癌ｃＤＮＡ文库含２７６×１０８重组子，克隆效率为３１９×１０１４重组子／ｇｃＤＮＡ。扩增后的文库浓度为４４×１０７重组子／μｌ，将文库稀释至１０－７时所产生的噬菌斑密度最为适宜。结论所构建的人前列腺癌高效ｃＤＮＡ文库为进一步筛选克隆前列腺癌的特异性癌基因或抗癌基因奠定了基础。     

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。     

青少年特发性脊柱侧弯椎间盘终板抑制消减cDNA文库的构建

用抑制消减杂交(SSH)构建组织间差异表达基因的cDNA消减文库，可进一步大批量筛选、克隆特发性脊柱侧弯表达的相关基因。     

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。     

丙型肝炎病毒 NS4A蛋白在胰腺细胞 cDNA文库中的结合蛋白基因的筛选

目的:从人胰腺细胞cDNA文库中筛选出与丙型肝炎病毒(HCV)NS4A蛋白存在相关作用并且参与糖、脂类代谢过程的结合蛋白,为研究HCV影响糖、脂类代谢的分子生物学机制提供实验依据.方法:构建pGBKT7-NS4A作为诱饵质粒,转化酵母菌株AH109,用SD/-Trp筛选阳性菌落.配合两种重组酵母菌株AH109与Y187...     

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.     

Purification and properties of gamma-glutamyl transpeptidase from human testis

Gamma-glutamyl transpeptidase was purified to apparent homogeneity from human testis by DEAE cellulose, acetone, precipitation, fractionation by ammonium sulphate, Sephacryl S-200 chromatography, Q-Sepharose chromatography and S-Sepharose chromatography following solubilization of the enzyme by Triton X-100. The purified enzyme had an apparent molecular weight of 54 KDa by Sephacryl S-200 gel filtration. On sodium dodecyl sulphate polyacrylamide gel electrophoresis, two submits of molecular weight 38 KDa and 14 KDa were obtained. The purified enzyme showed a single band with pI 6.0. The Km value and the optimal pH of the enzyme for L-gamma-glutamyl-3-carboxy-4-nitroanilide were found 1.09 mmol/l and 8.2-8.5, respectively. Serial lectin binding study with various lectin columns showed that the majority of the asparagine-linked oligosaccharides of the enzyme was complex-types. However, complex-types with bisecting N-acetylglucosamine residue were not recognized.     

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。