改良法克隆淋球菌特征核酸片段

目的：采用改良法克隆淋球菌特征核酸片段300bp到pBS.sk中,作为淋球菌检测试剂盒的阳性标本来源。方法：采用PCR扩增淋球菌特征核酸片段300bp,扩增条件：94℃30s,55℃1min,72℃1min,35个循环,另外用SmalI酶切,pBS,sk,与PCR产物采用改良法平端连接：连接温度14℃,72h,连接反应体积15μl,加10u SamlⅠ防止pBS.sk自连,连接完成后取5μl连接液转化200μlCaCl2制备的感受态受体菌JM109,用碱法提取重组子质粒,电泳比较质粒大小,PCR初筛,最后双酶切鉴定。结果：经过电泳比较质粒大小,PCR初筛,最后双酶切鉴定。结果：经过电泳比较质粒大小,PCR初筛,最后双酶切鉴定,得到5个克隆重组子。     

乙肝病毒X基因真核表达质粒及细胞模型的建立

采用聚合酶链反应（PCR）方法扩增HBVX基因,定向亚克隆入真核表达载体pRc／CMV2．构建重组质粒pCMV／X;采用脂质体法转染QSG7701细胞,筛选阳性细胞克隆;RT—PCR法检测HBV XmRNA的表达情况．Western blotting法鉴定HBx蛋白的表达,结果表明,构建的真核重组表达质粒pCMV／X中包含有完整的HBVX基因片断,pCMV／工转染的QSG7701细胞中有HBVXmRNA及HBx蛋白的稳定表达．成功建立了HBVX稳定转染的细胞系,为进一步探讨HBx在HCC的发生发展过程中所起的作用,提供了一个有价值的细胞模型．     

结核分枝杆菌分泌蛋白ESAT-6的表达、纯化和鉴定

目的：克隆结核分枝杆菌分泌蛋白ESAT-6基因,并在大肠杆菌中进行表达和纯化。方法：用PCR方法从结核分枝杆菌H37Rv基因组扩增出ESAT-6基因片段,克隆至pMD18一T载体中,序列测定正确后,将其亚克隆到表达载体pGEX-4T－1并在大肠杆菌DH5α中表达,表达蛋白经SDS—PAGE及Westem—blot分析后,亲和层析法纯化蛋白。结果：成功克隆了ESAT-6基因,并对其在E．coli中进行了表达,SDS—PAGE及Western—blot分析表明表达产物正确。通过GST纯化系统获得34kD纯化蛋白,与文献报道相符。结论：成功获得了纯化的ESAT-6蛋白,为进一步研究ESAT－6蛋白的致病机理提供了实验依据。     

双歧杆菌质粒聚合酶基因表达系统的构建及其鉴定

目的构建可以在双歧杆菌表达外源性基因的系统。方法PCR扩增双歧杆菌质粒聚合酶基因（BPP）并连接至质粒pBS—T以形成重组质粒pBS—BPP。PCR扩增双歧杆菌内源性阿拉伯糖苷酶的启动子及分泌性信号肽DNA序列（ara）并连接至质粒pBAD—A以形成重组质粒pBAD—ara,然后将增强绿色荧光蛋白（eGFP）基因连接至质粒pBAD—ara以形成重组质粒pBAD—ara—GFP。采用基因重组技术重组含有ara、BPP、eGFP基因序列并可将外源性基因分泌表达于菌体外及锚定表达于细胞壁的质粒pBS—BPP—ara—GFP,激光共聚焦显微镜下观察含有pBS—BPP—ara—GFP质粒及对照质粒的E．coli,验证eGFP定位表达情况。结果所构建的表达系统可以在E．coli中表达eGFP基因。结论通过基因重组方法成功构建了双歧杆菌表达系统,其可将外源基因分泌表达于菌体外。     

重组原核表达载体pQE30-HPV58L1的构建及鉴定

目的：构建重组原核表达载体以获得HPV58L1活性蛋白,为进一步研制HPV58基因工程疫苗打下基础。方法：用聚合酶链反应（PCR）扩增HPV58L1完整编码区基因,将PCR扩增产物克隆至pUC19质粒中并测序。利用pQE30质粒载体构建重组原核表达载体pQE30-HPV58L1,并通过酶切电泳验证重组结果的正确性。结果：PCR扩增出1.6Kb特异性片段,经克隆至pUC19后测序表明序列同源性与Gen-Bank报道一致。重组质粒pQE30-HPV58L1酶切后显示其大小约5.1Kb,酶切图谱与预期相同。结论：成功构建了重组原核表达载体pQE30-HPV58L1。     

CPSF在真核表达载体上的克隆与瞬时表达

目的：研究CPSF在真核表达载体上的克隆与瞬时表达。方法：以质粒pBS1761为模板扩增TAP-tag片段,PCR产物经纯化后克隆在真核表达载体pTRE2-hyg上。再以pUK-CPSF30k、73k、100k为模板扩增CPSF基因片段,将其克隆在质粒pTRE2-hyg-TAP-tag中TAP-tag片段的下游,并将重组质粒转化入细胞株Hela Tet-offS3细胞内。结果：细胞抽提液经SDS PAGE电泳后进行蛋白质印迹杂交,胶片上出现野生型的CPSF条带和分子量较大的滞后条带。后者经分子量与分子标记对照,确系重组体TAP-tag-CPSF所表达的蛋白条带。结论：重组质粒pTRE2hyg-TAP-tag-CPSF30k,73k,100k在Hela tet-offS3内完好表达。     

葡萄球菌A蛋白基因的克隆及其IgG受体区新型表达载体的构建

旨在克隆SPA基因并将该基因的IgG结合区亚克隆至毕赤酵母表达载体中。以金黄色葡萄球菌CowanI菌株基因组为模板,对葡萄球菌A蛋白(SPA)基因全长序列进行PCR扩增,再将PCR产物克隆入pMD18-T质粒,将DNA测序所得的结果用Blastn软件进行在线同源比对,经鉴定为SPA基因序列后,在线对其进行功能区域(IgG-Fc受体区)的预测,将该区域亚克隆入表达载体pPICZaA。结果显示,从CowanI菌株中成功地扩增到SPA基因,与NCBI中公布的序列同源性高达97%,同时构建了IgG-Fc受体区新型的表达载体     

一种利用ISSR开发SSR引物的方法

介绍从简单序列重复间区（ISSR）开发微卫星（SSR）引物的方法。该方法分两步：（1）正向引物的分离,即试材ISSR扩增,产物纯化回收并克隆至T载体,测序后设计正向引物（引物1）和巢式引物（引物2）;（2）反向引物的分离,即基医l组DNA酶切并连接接头,结合抑制PCR技术,对产物克隆测序后设计反向引物。应用该方法前人已在多个物种中开发出多对SSR引物．     

未知基因P2.18

用根据GRF设计的引物,以猪的下丘脑总RNA和mRNA为模板、进行RT—PCR扩增.将扩增产物纯化并克隆到T载体(pGEM3—Zf),自动测序仪测定其序列.发现所克隆的基因并非GRF基因.应用BLAST软件对其在INTERNET作了分析,未能确定为何种基因.     

人sTNFR1基因的克隆以及在NIH3T3细胞中的稳定表达

以He1a细胞的总RNA为模板,用RT—PCR方法扩增sTNFR1全编码区基因片段,构建含有目的片段的T载体克隆及真核表达载体pcDNA3．1（-）重组质粒亚克隆,将重组质粒和脂质体共同转染NIH3T3细胞系,G418筛选稳定转染细胞株．经核苷酸序列测序和酶切鉴定,成功构建了pcDNA3．1（-）-sTNFR1真核表达质粒,脂质体法建立了高效表达sTNFRI的稳定转染细胞系,并经RT—PCR和Western Blotting鉴定．人sTNFR1基因能在NIH3T3细胞系中稳定表达,为今后的研究打下了基础．     

Enzyme recruitment in vitro: use of cloned genes to extend the range of haloaromatics degraded by Pseudomonas sp. strain B13.

DNA fragments containing the xylD and xylL genes of TOL plasmid pWW0 -161 of Pseudomonas putida, which code for the catabolic enzymes toluate 1,2-dioxygenase and dihydrodihydroxybenzoic acid dehydrogenase, respectively, and the nahG gene of the NAH plasmid NAH7 , which codes for salicylate hydroxylase, were cloned in pBR322 vector plasmid. Deletion and insertion mutagenesis were used to localize these genes with respect to crucial endonuclease cleavage sites. The pBR322-based plasmids were ligated to the broad host range cloning vector pKT231 , or derivatives of it, and the hybrid plasmids were introduced into Pseudomonas sp. B13( WR1 ), a bacterium able to degrade 3-chlorobenzoate but not 4-chlorobenzoate, 3,5- dichlorobenzoate , salicylate, or chlorosalicylates . The cloned xylD gene expanded the catabolic range of WR1 to include 4-chlorobenzoate, whereas the cloned xylD - xylL genes enabled the isolation of derivatives of WR1 that degraded 3-chlorobenzoate, 4-chlorobenzoate, and 3,5- dichlorobenzoate . The cloned nahG gene extended the catabolic range of WR1 to include salicylate and 3-, 4-, and 5- chlorosalicylate .     

兽疫链球菌透明质酸分解酶基因的敲除

以透明质酸分解酶基因（Hyl）为改造目标, 利用同源重组技术获得 Hyl基因敲除的重组菌。首先以兽疫链球菌的基因组DNA为模板扩增出部分透明质酸分解酶基因（Hyl-1）,然后将其克隆到载体pMD19-T上,再以质粒pUC19为模板, PCR扩增得到氨苄青霉素抗性基因,通过反向PCR将其插入Hyl-1基因的中部,得到基因敲除载体pMD19T-SA。该载体与质粒pBR322分别用EcoRⅠ、PstⅠ双酶切后进行连接,得到基因敲除的重组载体pBR322 SA,PCR 及限制性酶切分析,构建的敲除载体与设计结果相符。最后,利用这两种敲除载体通过同源重组技术得到一株重组菌,经PCR 及酶活鉴定,这株菌的Hyl 基因已缺失。     

Highly efficient yeast-based in vivo DNA cloning of multiple DNA fragments and the simultaneous construction of yeast/ Escherichia coli shuttle vectors

In vivo recombinational cloning in yeast is a very efficient method. Until now, this method has been limited to experiments with yeast vectors because most animal, insect, and bacterial vectors lack yeast replication origins. We developed a new system to apply yeast-based in vivo cloning to vectors lacking yeast replication origins. Many cloning vectors are derived from the plasmid pBR322 and have a similar backbone that contains the ampicillin resistance gene and pBR322-derived replication origin for Escherichia coli. We constructed a helper plasmid pSUO that allows the in vivo conversion of a pBR322-derived vector to a yeast/E. coli shuttle vector through the use of this backbone sequence. The DNA fragment to be cloned is PCR-amplified with the addition of 40 bp of homology to a pBR322-derived vector. Cotransformation of linearized pSU0, the pBR322-derived vector, and a PCR-amplified DNA fragment, results in the conversion of the pBR322-derived vector into a yeast/E. coli shuttle vector carrying the DNA fragment of interest. Furthermore, this method is applicable to multifragment cloning, which is useful for the creation of fusion genes. Our method provides an alternative to traditional cloning methods.     

Stability of Integrated Plasmids in the Chromosome of Lactococcus lactis

Derivatives of plasmids pBR322, pUB110, pSC101, and pTB19, all containing an identical fragment of lactococcal chromosomal DNA, were integrated via a Campbell-like mechanism into the same chromosomal site of Lactococcus lactis MG1363, and the transformants were analyzed for the stability of the integrated plasmids. In all cases the erythromycin resistance gene of pE194 was used as a selectable marker. Transformants obtained by integration of the pBR322 derivatives contained a head-to-tail arrangement of several plasmid copies, which most likely was caused by integration of plasmid multimers. Single-copy integrations were obtained with the pSC101 and pTB19 derivatives. In all of these transformants no loss of the erythromycin gene was detected during growth for 100 generations in the absence of the antibiotic. In contrast, transformants containing integrated amplified plasmid copies of pUB110 derivatives were unstable under these conditions. Since pUB110 appeared to have replicative activity in L. lactis, we suggest that this activity destabilized the amplified structures in L. lactis.     

Construction of cell lines that regulate by temperature the amplification and expression of influenza virus non-structural protein genes.

Monkey cell lines have been transformed with a mixture of plasmids pSV2neo and pSLVa232N, a derivative of plasmid pSLVa232 (Portela et al., 1985b). Plasmid pSLVa232N contained the influenza virus genes encoding non-structural proteins under the control of the SV40 late promoter in pSLts1 vector that includes the SV40 ori and the tsA209 T-antigen gene. At restrictive temperature, plasmid sequences remained stably integrated in the cell genome, but upon temperature shift-down, defined circular DNA molecules were generated and amplified up to 2000-5000 copies/cell. Restriction analysis, Southern blot hybridization and partial sequencing indicate that one such episome, pC5, was derived from the integrated plasmid sequences by a homologous recombination event that led to deletion of the pBR322 sequences included in pSLVa232N. Concomitant with gene amplification, an induction of 20-65-fold in the expression of NS1 and NS2 proteins was observed after temperature shift-down. Thus, gene cloning into vector pSLts1 and transformation at restrictive temperature of cells permissive for SV40 DNA replication, appears to be a useful strategy for the controlled amplification and expression of cloned genes.     

Cloning and characterization of a plasmid DNA from anacystis nidulans 6301

A plasmid DNA of Anacystis nidulans 6301 was isolated by CsCl-EtBr centrifugation. The Mr of the plasmid, named pBA1, was estimated to be 5.04 +/- 0.26 X 10(6) by electron microscopic analysis and 5.2 X 10(6) by agarose gel electrophoresis. The pBA1 DNA was opened at a unique site with BamHI and cloned in pBR322 vector propagated in Escherichia coli HB101 cells. The recombinant plasmid, named pBAS18, was digested with various restriction endonucleases and its cleavage map was constructed. Based on this result, the cleavage map of the pBA1 plasmid is presented.     

The use of a partition locus to increase stability of tryptophan-operon-bearing plasmids in Escherichia coli

The stability of different derivatives of plasmid vectors pBR322 and pACYC184 carrying the tryptophan operon of Escherichia coli was monitored in various media. It was found that in the absence of any special selective pressure, all plasmids were lost from the culture. The stability varied depending both on the orientation of the inserted tryptophan fragment and the growth media used. The pBR322::trp+ plasmids were lost at an average frequency of 0.3 to 0.8% per cell generation, while the pACYC184::trp+ plasmid was lost at a rate higher than 5%. In all cases the whole plasmid was lost at a rate higher than 5%. In all cases the whole plasmid was lost, indicating a high stability of the plasmid::cloned DNA as such. To increase the stability of the cloning vectors, the partition locus of plasmid pSC101 was added to both the pBR322::trp+ and pACYC184::trp+ plasmids. The addition of this gene increased the replicon stability at least 3- to 10-fold, with the pBR322::trp+-par+ plasmids being the most stable. Also in this case, the stability was dependent on the plasmid type and on the growth medium. In no case was there a discoordinate loss of the antibiotic-resistance and tryptophan genes from the vectors.