质粒

Cloning of the Schwanniomyces occidentalis α-amylase and high expression in S.cerevisiae.The E.coli / yeast shuttle plasmid YCEpl partial library of Schwanniomyces occidentalis DNA was constructed and α-amylase gene fragments were screened in Saccharomyces cerevisiae by amylolytic activity.Several transformants with amylolysis were obtained and one of the fusion plasmids has about 5.0 kb inserted DNA fragment.It contains the upstream and downstream sequences of α-amylase gene from S.occidentalis .

以E.coli/yeast穿梭质粒YCEp1为载体构建西方许旺酵母部分基因组文库，在大肠杆菌中扩增后提取混合质粒DNA，经电转化非缺陷标志酒精酵母 AS.2.1364，在YPDS平板(含1%葡萄糖和1%可溶性淀粉)上用淀粉水解活力筛选含水解淀粉的阳性转化子，从阳性转化子中分离重组质粒证实含5.0kb的插入片段，用α-淀粉酶基因两端序列设计的引物PCR扩增及扩增片段序列分析证实该片段中含有α-淀粉酶全部编码序列。

After the NK gene from recombinant plasmid pUC19-NK was cloned on expression vector pBV220, it was transformed into host cell E. coli HB101. Recombinant plasmid pBV220-NK was extracted for identifying by analysis of restriction enzymes, PCR and sequencing. Studies on recombinant strain's growth and expression showed that heterogene have no distinct effect on host cell. The recombinant plasmid have excellent segregational stability but low structural stability. The results of kinetics of pBV220-NK expression in E.

将重组质粒pUC19-NK上的纳豆激酶基因成功克隆到表达载体pBV220上，并将之转入大肠杆菌HB101中，得到转纳豆激酶基因工程菌，提取重组质粒经单酶切、双酶切及PCR分析验证后，对重组菌的生长及表达进行一系列研究，结果表明：外源纳豆激酶基因的导入对宿主的生长没有明显的影响：重组质粒的稳定性实验表明：该质粒具有良好的分离稳定性，而结构稳定性较差。

The PSCA_3 fragment was selected for its superior expression level in eukaryotic cells.Then the sig-PSCA_3-Fc-GPI genetic fragment was cloned into pVAX1-neo-IRES-GM/B7 vector to construct the final immunological inhanced DNA vaccine pVAX1-PSCA_3-FcGB. Immunofluorescence and flow cytometry were used to confirm the expression of PSCA_3 fragment by transfected into Cos7 cell.Finally,the anti-tumor effect of pVAX1-PSCA_3-FcGB was tested in murine prostate cancer model generated by RM-1 cell line.The animal was immunized with pVAX1-PSCA_3-FcGB DNA vaccine by intramuscular injection plus electroporation,pVAX1 and pVAX1-PSCA_1-FcGB plasmid were used as control.The inhibitory effect of tumor was investigated by observion of forming time,volume and inhibition ratio of tumor.Results:DNA sequencing conformed that the heterological PSCA fusion antigen fragment which was synchronized by overlapping-extending-PCR,was consistent to design.Enzyme digestion analysis showed that the 1 to 4 copies heterological PSCA fusion antigen fragments were constructed successfully.

方法(1)检索GenBank，选择包含人主要T细胞抗原表位序列的人PSCA基因片段，应用异种化抗原设计技术，保留人T细胞抗原表位，设计异种化PSCA融合抗原片段；(2)根据核酸序列按中心模板法设计引物，应用重叠延伸PCR技术拼接合成异种化PSCA融合抗原片段基因，以PCR、限制性酶切和DNA序列测定法进行鉴定：(3)利用DNA限制性内切酶BssHⅡ和MluⅠ酶切后粘端互补的特点，采用同尾酶法构建1—4拷贝异种化PSCA融合抗原片段(PNCA_1-PSCA_4)，并将上述片段分别插入真核表达载体pCI-neo-Fc-GPI中，转染293T细胞，借助免疫荧光+流式细胞术考察插入片段表达效率，最终选定PSCA_3片段进行下一步研究；(4)将sig-PSCA_3-Fc-GPI基因片段自pCI-PSCA_3-Fc-GPI质粒上切下，插入pVAX1-neo-IRES—GM/B7载体中，构建免疫增效DNA疫苗pVAX1-PSCA_3-FcGB，并应用转染Cos7细胞+免疫荧光/流式细胞术方法鉴定其在真核细胞中的表达情况；(5)给8周龄雄性C57BL/6小鼠皮下种植RM-1细胞，制备小鼠前列腺癌模型，并采用股四头肌肌肉注射+电脉冲法(Electroporation，EP)接种DNA疫苗质粒pVAX1-PSCA_3-FcGB，同时接种pVAX1空载体质粒和pVAX1-PSCA_1-FcGB质粒作为对照，通过观察计算免疫动物的成瘤时间、肿瘤体积和抑瘤率，来评价该DNA疫苗在小鼠体内的抑瘤效果。

Adult rats are divided into control group, intrapericardial injection group and negative group, sublingual vein injection group and negative group. 6ds after transfection, issues of heart, liver, lung and kidney are stained with X-gal to observe transfection to myocardium and non-target transfection. 3. CaN Aβ gene silencing in vivo. Adult rats are divided into Control group, hypertrophy model group, intrapericardial injection group and negative group, sublingual vein injection group and negative group.

二整体动物水平上探讨心肌有效转染途径：以PLacZ为报告基因，将成年大鼠分为空白对照纽(生理盐水心包腔内注射后超声导入)；转染质粒心包腔内注射组及其阴性对照组：质粒+微泡+酶的混合物心包腔内注射，超声导入；转染质粒舌下静脉注射组及其阴性对照组：质粒+微泡混合液经舌下静脉注射，超声导入。

Formation of mating pairs, nicking of the F oriT sequence, and transfer of the 5' end of a single strand of F DNA proceed as in transfer of the F plasmid. Therefore, Part of the plasmid is transferred first, Chromosomal genes are transferred next, and the rest of the plasmid is transferred last.

雌雄菌的交配、F质粒oriT处缺口的形成、以及由单链F DNA5'末端开始转移的过程都与F质粒的转移相同，因此，首先转移的是部分质粒片段，其次是染色体基因，剩余的质粒片段最后被转移。

One could nodulate the soybean cultivars tested and showed wider host range, the other only nodulate limited soybean cultivars. Plasmid could not be detected from twenty-three slow-growing strains and twenty-nine fast-growing strains were found containing 1-4 plasmids by Echardts method.

质粒快速检测结果表明：慢生型大豆根瘤菌均不含有质粒，快生型大豆根瘤菌均有1-4个质粒，质粒类型与宿主亲和性也有一定的相关性。

Chicken IL-15(ChIL-15), Which was discovered in 2001, plays a main role in activating NK cells and CD8+ memory T cells, and may therefore have potential as a vaccine adjuvant.According to published ChIL-15 gene sequence, a pair of primers were designed and synthesized to clone ChIL-15 cDNA from ConA-activated chicken splenocytes by RT-PCR. Recombinant plasmid pUC19ChIL-15 carrying the ChIL-15 gene was constructed. The gene encoding mature ChIL-15 (mChIL-15) was amplified from pUC19ChIL-15 by PCR and cloned into pMD 18-T vector. After digested with Kpn I /Pst I , the mChIL-15 gene was subcloned into prokaryotic expression vector pPRoEX橦Ta and transformed into E.coli DH5a .The transformant was induced by IPTG, and the recombinant ChIL-15(rChIL-15) was expressed as a fusion protein with a histidine hexamer tag at the N-terminal end of the protein. Following expression, the protein was purified by ProBond?Nickel-Chelating Resin. The uninduced and induced protein lysates and the purified protein were separated by SDS-PAGE and transferred onto nitrocellulose membrane to identify rChIL-15 by Western blot. The rChIL-15 was used to immune the guinea pig to obtain rChIL-15 polyclonal antibody.

参考已发表的ChIL-15基因序列，设计合成引物，应用RT-PCR技术从刀豆球蛋白A活化的白来航鸡脾淋巴细胞中克隆ChIL-15 cDNA，将其与SmalⅠ酶切处理的pUC19载体连接，构建重组质粒pUC19ChIL-15；用PCR技术从pUC19ChIL-15中扩增出去信号肽的成熟ChIL-15(mChIL-15)基因，与pMD 18-T载体相连构建重组质粒pMDChIL-15；然后用KpnⅠ／PstⅠ双酶切下mChIL-15基因片段，定向亚克隆到经同样酶切处理的带有6个组氨酸标签的表达载体pP_RoEX~HTa中，构建重组质粒pP_RoChIL-15，对其测序确认读框正确后，转入大肠杆菌DH5α中诱导表达并进行纯化，用重组ChIL-15(rChIL-15)免疫豚鼠，制备多克隆抗体；再用HindⅢ／PstⅠ从质粒pP_RoChIL-15上切下mChIL-15基因，定向亚克隆到经同样酶切处理的杆状病毒转移载体pMelBacB中，经酶切、PCR和序列测定鉴定后，与杆状病毒线形化DNABac-N-Blue~(TM DNA共转染Sf9昆虫细胞，经三轮蚀斑纯化，构建重组杆状病毒rBac-ChIL-15，用该重组病毒感染处于对数生长期的Sf9细胞，按不同的时间收取样品，离心后对其上清和沉淀进行SDS-PAGE和Western blot分析。

By means of experiments of marker transfer,acridine orange sensitivity test,F' curring and mini-chromosome transformation it is.conchi-ded that the F' plasmid is always in an integrated state in the Sin strains and that the initiation of the replication of the bacterial chromosome is carrie...

标记转移、吖啶橙敏感性，F′质粒消除和mini-染色体质粒转化等实验说明，Sin菌株中F′质粒始终处于整合状态，并且在40℃中细菌染色体的复制由整合状态的F′质粒所带动。

By means of experim ents of marker transfer,acridine orange sensitivity test,F' curring and mini-chr omosome transformation it is concluded that the F' plasmid is always in an integ rated state in the SIn strains and that the initiation of the replication of the bacterial chromosome is carried on by the integrated F' plasmid.

标记转移、吖啶橙敏感性、F′质粒消除和mini-染色体质粒转化等实验说明，Sin菌株中F′质粒始终处于整合状态，并且在40℃中细菌染色体的复制由整合状态的F′质粒所带动。

A process for preparing the recombinant chicken gamma-interferon (rCHIFN-gamma) with high activirus activity includes such steps as cloning ChIFN-gamma gene from eukaryotic plasmid to transfer carrier by dual enzyme servenings to obtain recombinant transfer plasmid pFASTBACI-ChIFN-gamma, taking DH10Bac competent cell, adding IngpFASTBAC1-ChIFN-gamma, transposition, diluting to culture object by SOD culture medium, coating on Luria plate, culturing, choosing white clony, purifying, naming positive plasmid as Bacmid-ChIFN-gamma, extracting its DNA, and transfecting Sf9 cells.

高抗病毒活性重组鸡γ-干扰素的制取方法及用途，涉及一种广谱高效抗病毒基因工程产品的制取方法。将ChIFN-γ基因从真核质粒中经双酶切后克隆到转移载体中，获得重组转移质粒pFASTBAC1-ChIFN-γ；取DH10Bac感受态细胞，加入1ng pFASTBAC1-ChIFN-γ，转座后，用SOC培养基稀释培养物，涂布Luria平板，培养后，挑取白色菌落，Luria平板进行纯化，阳性质粒命名为Bacmid-ChIFN-γ，提取阳性质粒DNA；取上述重组DNA质粒转染Sf9细胞，制得高抗病毒活性重组鸡γ-干扰素。

She gently rebuff ed him, but agreed that they could be friends

她婉言拒绝了，但同意作为朋友相处。

If in the penal farm, you were sure to be criticized.

要是在劳改农场，你等着挨绳子吧！