克隆

In our study,two important lepidopteron pests,beet armyworm and cotton bollworm were surveyed for their endogenous piggyBac.As a result,endogenous piggyBac elements were found in these two insects,and one of the piggyBac elements isolated from the genome of cotton bollworm was intact and thought to be potentially active.1 Clonging and sequence analysis of piggyBac from beet armywormUsing PCR technique,with degenerate primers,a DNA fragment of piggyBac-like element was cloned from the genome of beet armyworm,spodoptera exigua hubner.The DNA fragment was 456bp in length,and the deduced amino acid sequence shares 50%- 78% similarity with other piggyBac elements from insects.But one stop code was found in this DNA fragment.

由于转座子很难用常规的RT-PCR和RACE技术进行克隆鉴定，故本研究在兼并引物PCR的基础上，利用inverse PCR和vectorret PCR技术，不仅调查了鳞翅目两种重要的农业害虫甜菜夜蛾和棉铃虫的內源piggyBac存在情况，同时克隆到了內源性的piggyBac转座子，并且从棉铃虫中获得了一个结构完整、具有潜在活性的piggyBac转座子HaPLE1.1甜菜夜蛾piggyBac转座子基因的克隆与序列分析采用PCR技术，利用兼并引物，从甜菜夜蛾基因组中克隆出一个內源性piggyBac类似因子的DNA片段，并命名为SePLE。

In this experiment, we screen the major protective antigen gene-SOD gene of M. paratuberculosisin order to study the sensitive, specific diagnostic reagent and prophylaxis preparation, especially theDNA vaccine. The SOD gene was amplified from Mycobacterium paratuberculosis C-2 chromosomalDNA by using the PCR technique and cloned into pMD18-T Vector System. We gained a SOD gene of624bp.The recombinant clone was identified byα-complementarity, enzyme digestion and PCRidentification. The result indicated that the recombinant plasmid pMD18-T-SOD was successfullyconstructed. Moreover, through sequential determination and DNASTAR analysis between the clonedSOD gene of M. paratuberculosis C-2 and that of the M.paratuberculosis K-10 strain, the sequentialhomogeneity reached 99%, and the amino acid homogeneity reached 99.5%. The preceding analysisindicated that the SOD gene was very conservative in M. paratuberculosis.

为了研制副结核病敏感、特异的诊断试剂和新型、高效的预防制剂，尤其是DNA疫苗，本研究筛选了M.paratuberculosis主要保护性抗原SOD基因，以M.paratuberculosis C-2染色体DNA为模板，以SOD基因的特异性引物进行PCR扩增，获得了624bp的SOD基因，通过T-A克隆技术，将PCR产物克隆至pMD18-T Vector中，以质粒大小、酶切分析、PCR扩增及序列分析鉴定重组克隆，成功地构建出克隆质粒pMD18-T-SOD，序列测定及DNASTAR分析表明，所获得的M.paratuberculosis C-2 SOD基因与Gen Bank中M.paratuberculosis K-10 SOD基因的大小完全一致，两者核苷酸序列的同源性为99％，氨基酸序列的同源性为99.5％，表明该基因在副结核分枝杆菌中是高度保守的。

Another two field container experiments were conducted to study intraclonal spatial division of labour in Fragaria vesca and Lysimachia christinae with different branching types and Lysimachia christinae at different altitudes in response to reciprocal patchiness of light and nutrients.

结果显示：克隆整合显著增强蛇莓和野草莓胁迫分株的生长，耗-益分析表明对未受胁迫分株没有显著损耗，故整个克隆片段的生长得到显著提高，克隆整合对于克隆植物在高山环境中的种群扩展、生境开拓可能是非常重要的。

Three kinds of embryos' chromosome, including goat fertilization embryo, goat-goat cloned embryo and goat-rabbit cloned embryo were analyzed, The results showed that all of them have 60 chromosomes with same character, all were telocentric chromosome, besides the X-chromosome is the second larger telocentric chromosome.

经对超排获得的山羊受精胚、山羊同种克隆胚和羊-兔异种克隆胚的囊胚期胚胎染色体分析表明，三种来源的胚胎其染色体数目均为60条，且所有常染色体均为端部着丝点染色体，X染色体为第二大的端部着丝点染色体，说明山羊同种克隆胚和山羊-兔异种克隆胚均为山羊的核型，是供体细胞核在不同胞质中的重新编程的结果。4。

Temperature, clone and their interaction influenced significantly the neonate starvation, and body, egg and neonate volumes in the offspring of B. calyciflorus. The neonate starvation times of all the thirteen clones were curvilinearly correlated with temperature. The neonate starvation times of Clone SP1 was negatively correlated with body volumes. The neonate starvation times of Clone SP2 was negatively correlated with neonate volumes, but that of Clone SU1, SU3 and SU7 were positively correlated with neonate volumes.

回归和偏相关分析表明，所有克隆的幼体耐饥饿时间均与温度间呈曲线相关；克隆SP1的幼体耐饥饿时间与母体体积间存在显著的负相关关系；克隆SP2幼体耐饥饿时间与幼体体积间有显著的负相关关系，克隆SU1、SU3和SU7的幼体耐饥饿时间与幼体体积间均有显著的正相关关系。

Restrictive changes of TCR Vp repertoire and clonal expanded T cells could be found in peripheral blood T cells from patients with T-ALL. However, the clonal expanded T cells' property (clonal expansion leukemic cells or leukemia antigen-specific expansion T cells) should be further characterized. It may play a certain role on detection of minimal residual disease and design of anti-leukemia idiotypic vaccine. The dominant utilization of TCR VjJ repertoire could be found in peripheral blood T cells from patients with B-ALL, meanwhile clonal expansive T cells were existed. It may be a feature of the host immune response for leukemia-associated antigen. The clonal expansive tendency of T cells in V021 and VP23 subfamilies was rather obvious, which may be correlated with certain malignant B-cell clone. The CDR3 sequences of monoclonal expansive T cell in T cell strains were different, which was the base of developing DNA vaccine.

T-ALL患者外周血T细胞的TCR Vβ谱系出现限制性改变，均可检测到克隆性增殖T细胞，尚需进一步鉴定其性质(肿瘤性或抗原特异性增殖)，对于研究微小残留病变检测和设计抗白血病独特型疫苗均有一定的意义。B-ALL病人外周血T细胞的TCR Vβ谱系呈现优势利用的特点，并存在克隆性增殖的T细胞，这可能是机体对白血病相关抗原产生的特异性免疫反应；Vβ21和Vβ23亚家族T细胞发生克隆增殖改变的趋向性比较明显，可能与B-ALL相关抗原刺激有关。T细胞株中检测到的肿瘤克隆其CDR3序列具有高度的特异性，是构建DNA疫苗的基础。

DGGE result showed that As-I1-3 was dominant in arsenite enriched community, followed by isolate As-I1-5 (Halomonas meridiana,100%) and Mn-I1-6 (Marinobacter vinifirmus, 99%). 16S rDNA library analysis reconfirmed the result of DGGE, which showed that the three bacteria occupied 72.5%, 10% and 7.5% of the total OTUs, respectively.

DGGE分析显示，该菌是富集物中的最优势菌，其次是盐单胞菌和海杆菌。16S rDNA 克隆文库分析结果进一步证明，与菌株AS-I1-3序列相同的克隆子占整个克隆文库的72.5%；而与菌株AS-I1-5 (Halomonas meridiana， 100%)和菌株Mn-I1-6（Marinobacter vinifirmus，99%）序相同的克隆子分别占10%和7.5%。

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。

The PCR products were examined by agarose gel electrophoresis. The target gene fragments were purified by gel extraction kit and ligated to cloning vector pMD18-T. The recombinant vectors were transformed into host strain E. coli K802 by lithium chloride method, screened and identified with PCR and restrictive enzymatic digestion. Their sequences were confirmed by DNA sequencing.(2) sTWEAK1 gene was subcloned into expression vector pProEx HTb and transformed into E. coli BL21. sTWEAK2 gene was subcloned into expression vector pMAL-C2x and transformed into E. coli TB1. The recombinant vectors were screened and identified with PCR and restrictive enzymatic digestion. The recombinant fusion proteins were induced to express with IPTG, detected by coomassie brilliant blue-stained SDS-polyacrylamide gel electrophoresis , and confirmed by Western blot analysis.(3) The sTWEAK1 fusion protein was purified with Ni-NTA Spin Kit.(4) The biological activity was assayed on transformed and tumor cells by microplate photometer after crystal violet or sulfur rodamine B staining.(5) The contents of IL-8 in the supernatant of 1990 cell cultures were determined by ELISA.(6) The morphological changes of the sensitive cells were observed by light and transmission electron microscopies.(7) The cell cycle and apoptotic rate were assayed by flow cytometry in 1990 and M85 cells.(8) The effect of fusion proteins on induction of NF-κB in 1990 and LOVO cells was detected with Dual-Luciferase Reporter Assay system.(9) The TWEAK gene was subcloned into Adeno-X Viral DNA with pShuttle vector and transfected into HEK293 cells by lipofectamine method.

(1)本研究用RT-PCR方法，从人组织细胞总RNA中扩增可溶性TWEAK胞外区（sTWEAK1和sTWEAK2）的cDNA序列及全长编码序列，用琼脂糖凝胶电泳分析PCR产物，胶回收目的基因片段，连接到pMD18-T克隆载体中，转化大肠杆菌K802，PCR和酶切筛选阳性克隆，全自动DNA测序验证序列；(2)sTWEAK1和sTWEAK2分别亚克隆到pProEx HTb和pMAL-C2x表达载体中，分别转化大肠杆菌BL21和TB1，PCR筛选和酶切鉴定，阳性克隆用IPTG诱导表达，表达产物用SDS-PAGE分析和Western blot验证融合蛋白；(3)用NTA-Ni Spin试剂盒初步分离纯化sTWEAK1融合蛋白；(4)用体外培养的肿瘤细胞和正常对表达产物进行活性检测，贴壁细胞用结晶紫染色法，悬浮细胞用磺酰罗丹明B染色法，酶标仪检测OD值；(5)敏感细胞用ELISA法检测细胞培养上清中IL-8的含量；(6)用光镜和电镜观察敏感细胞死亡和细胞凋亡情况；(7)用流式细胞仪分析表达产物对敏感细胞凋亡率和细胞周期的影响；(8)用双荧光素酶报告基因检测法，测定表达产物对敏感细胞NF-κB的影响；(9)用pShuttle穿梭质粒将TWEAK重组到腺病毒载体上，用脂质体转染法转染HEK293细胞，PCR鉴定重组质粒。

The pHybLex/Zeo-Idl plasmid and the cDNA library plasmid were sequentially transformed into the yeast swains and screened to obtain Leu2^＋ and Leu2^＋LacZ^＋ clones, with the false positive clones excluded using positive and negative controls, and the plasmid of the true positive clone was sequenced and blasted for homological analysis.

方法PCR方法扩增Id1的编码序列并定向克隆入诱饵质粒pHybLex/Zeo，构建重组诱饵质粒pHybLex/Zeo-Id1，酶切鉴定后转化入酵母株EGY48/pSH18-34，检测重组诱饵质粒有无非特异激活报告基因Leu2、LacZ作用；扩增并提取成人肺组织文库质粒，将文库质粒及重组诱饵质粒转化入酵母细胞，依次筛选Leu2^＋，Leu2^＋LacZ^＋克隆；设置阴性及阳性对照，重复筛选Leu2＋LacZ＋克隆并排除假阳性克隆，获取真阳性文库质粒，进行测序和同源性比对。

With Death guitarist Schuldiner adopting vocal duties, the band made a major impact on the scene.

随着死亡的吉他手Schuldiner接受主唱的职务，乐队在现实中树立了重要的影响。

But he could still end up breakfasting on Swiss-government issue muesli because all six are accused of nicking around 45 million pounds they should have paid to FIFA.

不过他最后仍有可能沦为瑞士政府&议事餐桌&上的一道早餐，因为这所有六个人都被指控把本应支付给国际足联的大约4500万英镑骗了个精光。