共查询到20条相似文献,搜索用时 527 毫秒
1.
2.
3.
目的:应用AdEasy-1 系统构建包含tmTNF-alpha单克隆抗体轻、重链序列的重组腺病毒表达载体。方法:首先PCR 合成抗体
轻、重链序列,分别将轻、重链序列插入经过改造的含有双启动子的穿梭质粒pShuttle-2CMV,将穿梭载体电转化转化AdEasy 系
统BJ5183 感受态,挑取单克隆扩增质粒后酶切鉴定。结果:成功构建重组腺病毒表达载体pAdEasy-tmTNF-alpha,抗体重链、轻链序
列酶切后经1%琼脂糖凝胶电泳证实条带片段大小正确,电转化BJ5183 后挑选重组克隆提取质粒,PacI酶切后重组片段位于4.5
kb及3 kb位置,证明重组腺病毒质粒pAdeasy-1-tmTNF-alpha构建成功。结论:将腺病毒系统与单克隆抗体技术相结合,利用AdEasy-1
系统成功构建腺病毒重组tmTNF-alpha单克隆抗体表达载体,为进一步开展肿瘤基因治疗的研究提供基础。 相似文献
4.
5.
碳酸钙沉淀法回收琼脂糖凝胶中DNA的探讨 总被引:5,自引:2,他引:3
采用碳酸钙沉淀法回收琼脂糖凝胶中的DNA,达到分离纯化目的,回收后的DNA可用于重组、PCR等研究。首先将含有目的DNA的琼脂糖凝胶用Nal溶液融解,然后加入cacl2,和NaHCO3,生成CaCO3,沉淀,DNA与cac03形成复合物,通过离心分离出沉淀复合物,利用稀酸溶解沉淀,再用无水乙醇沉降,即可回收目标DNA。利用该方法回收了质粒、毛白杨和转基因羊基因组DNA,同收率为20%~50%,0D260/OD280,为1.7~19,最大回收了21kb片段,最小回收250bp片段,回收后的DNA样品进行了PCR扩增和限制性内切酶反应,PCR可以扩增出目的片段,同时限制性内切酶可以将回收后的DNA切开,表明DNA质量良好。利用碳酸钙沉淀法可以回收琼脂糖凝胶中的DNA,此法简单、易行,较为有效。 相似文献
6.
《现代生物医学进展》2014,(22)
目的:应用AdEasy-1系统构建包含tmTNF-α单克隆抗体轻、重链序列的重组腺病毒表达载体。方法:首先PCR合成抗体轻、重链序列,分别将轻、重链序列插入经过改造的含有双启动子的穿梭质粒pShuttle-2CMV,将穿梭载体电转化转化AdEasy系统BJ5183感受态,挑取单克隆扩增质粒后酶切鉴定。结果:成功构建重组腺病毒表达载体pAdEasy-tmTNF-α,抗体重链、轻链序列酶切后经1%琼脂糖凝胶电泳证实条带片段大小正确,电转化BJ5183后挑选重组克隆提取质粒,PacI酶切后重组片段位于4.5kb及3 kb位置,证明重组腺病毒质粒pAdeasy-1-tmTNF-α构建成功。结论:将腺病毒系统与单克隆抗体技术相结合,利用AdEasy-1系统成功构建腺病毒重组tmTNF-α单克隆抗体表达载体,为进一步开展肿瘤基因治疗的研究提供基础。 相似文献
7.
目的:制备一种具有琼脂糖凝胶电泳定量功能的DNA分子量标准。方法:以pMD18-TSimple载体为基础骨架构建了长度为4.7kb的质粒,应用定点突变的方法,在载体上分别间隔100bp、200bp、400bp、800bp、1200bp处,加入了HindⅢ限制性内切酶的酶切位点,将该质粒扩增后并应用HindⅢ酶切后,1.5%琼脂糖凝胶电泳鉴定。结果:获得的分子量条带大小依次为100bp、200bp、400bp、800bp、1200bp和2000bp,每次使用4μl可获得质量范围为10ng、20ng、40ng、80ng、120ng和200ng的定量标准品。结论:应用该方法制备标准品,具有制备简单、成本低、定量快速等优点。 相似文献
8.
本文介绍一种从重组质粒中快速提纯DNA插入片段的方法。质粒DNA的制备简单、快速、分离的质粒DNA可用于限制性酶切和转化大肠杆菌等。从琼脂糖凝胶中提纯DNA插入片段的方法操作简单,回收效率高,提纯的DNA片段可用于连接和制备杂交探针等。 相似文献
9.
目的:发展一种简单快速经济的回收琼脂糖凝胶中DNA的方法.方法:将切的琼脂糖凝胶胶块放入嵌套Eppendorf管中捣碎,加入50μL机油,室温下12 000r/min离心5min,取大Eppendorf管中收集的液体跑胶检验,凝皎上包括代表100bp~2000bp不同大小DNA分子回收率的分子量标准DL2000.结果:DNA回收率可由加机油前的约35%提高为加机油后的45%-90%,回收效率的波动主要取决于DNA片段的大小、切下的含有DNA的胶块的大小及操作者的熟练程度.结论:该方法快速、简便、经济,具有良好的重复性与特异性,比许多国产的试剂盒更可靠. 相似文献
10.
PCR特异产物回收纯化方法的比较 总被引:1,自引:1,他引:0
方法:采用三种方法对苹果褪绿叶斑病毒RT-PCR的特异DNA产物进行回收纯化。目的:针对不同情况,选择适宜的回收纯化方法。结果:用普通琼脂糖替代低融点琼脂糖,回收纯化后产物的浓度及纯度与低融点琼脂糖法基本一致,完全可以用普通琼脂糖替代低融点琼脂糖进行DNA片段的回收纯化,从而降低成本,简化操作。玻璃奶法的回收纯度明显高于低融点琼脂糖法和普通琼脂糖法,且更快速安全,是采用普通琼脂糖法还是采用玻璃奶法回收纯化DAN片段应以实际需要而定。 相似文献
11.
目的:扩增先天性免疫中具有重要功能的泛素连接酶TRIM25及其不同结构域的cDNA,构建带有不同标签的融合蛋白载体并进行细胞表达。方法:以TRIM25 cDNA为模板,PCR扩增不同结构域cDNA,扩增产物及载体经酶切、连接后,转化大肠杆菌DH5α,挑克隆,提取重组质粒后酶切鉴定、测序,将测序正确的重组质粒转染293细胞,用Western印迹对融合蛋白的表达进行鉴定。结果:TRIM25、Two-BOX结构域、SPRY结构域以正确读框插入Flag-pcDNA3.0,TRIM25以正确读框插入pCMV-Myc,RING结构域、CDD结构域、Two-BOX结构域以正确读框插入pEGFP-c1,上述重组质粒能够在293细胞中表达。结论:构建了TRIM25及其突变体的重组表达质粒并获得表达,为研究不同RNA病毒通过与TRIM25作用抑制宿主功能提供了基础。 相似文献
12.
Khalil Amjad B. Anfoka Ghandi H. Bdour Salwa 《World journal of microbiology & biotechnology》2003,19(3):239-241
The plasmid profile of two thermophilic bacterial strains isolated from recreation thermal springs in Jordan has been investigated. These strains are Streptococcus thermophilus and Bacillus sp1, which have been isolated from Zerka – Maeen and Himma hot springs respectively. Supercoiled and circular plasmid forms were detected, explaining the effect of DNA conformation on the mobility of the plasmid in the agarose gel electrophoresis. Two plasmids have been isolated and characterized by restriction endonucleases to facilitate their use as cloning vectors in thermophilic strains. The sizes of the plasmids were approximately 3 kb (from Streptococcus thermophilus) and 7 kb (from Bacillus spl). These plasmids were then digested with three different restriction enzymes (EcoRI, Bam HI, and HindIII), one of which was found to possess a single site for both plasmids, this enzyme is EcoRI. 相似文献
13.
Summary A rapid, simple, and sensitive method for plasmid copy number comparison was developed. The extracted plasmids from the same
amount of cells were subjected to agarose gel electrophoresis and the gels photographed. The photographs were processed by
a Macintosh image analyser to enumerate the densities of plasmid bands. As a size reference, λ-DNA digested with a restriction
enzyme was used. The densities divided by size of plasmids (base pair) would represent relative values of their copy numbers. 相似文献
14.
Improved lysis of group N streptococci for isolation and rapid characterization of plasmid deoxyribonucleic acid. 总被引:31,自引:0,他引:31
Procedures for effective cellular lysis and plasmid deoxyribonucleic acid (DNA) isolation from group N streptococci were developed. Cells were grown at 32 degrees C for 4 h in a modified Elliker broth containing 20 mM DL-threonine. After cellular digestion with 2 mg of lysozyme per ml for 7 min at 37 degrees C, 1% sodium dodecyl sulfate exposure resulted in complete and immediate lysis. Lactose (Lac) plasmid species in Streptococcus lactis C2 and S. cremoris B1 (30 and 37 megadaltons, respectively) were demonstrated upon examination of DNA from the cleared lysates by agarose gel electrophoresis. Increasing the lysozyme treatment to 20 min or more resulted in loss of the Lac plasmid, whereas other resident plasmids were unaffected and demonstrable in agarose gels. Diethylpyrocarbonate added before lysis prevented Lac plasmid loss in 20-min lysozyme-treated cells, but was not effective after 40 min of lysozyme treatment. The results suggested that endogenous nuclease activity during the lysozyme treatment period initiated Lac plasmid DNA loss. The development of an efficient lysis procedure for the group N streptococci allowed rapid identification and characterization of plasmid DNA by agarose gel electrophoresis. The plasmid composition of S. lactis C2 and S. cremoris B1, as determined by agarose gel electrophoresis, compared favorably to previous electron microscopic observations. 相似文献
15.
Improved lysis of group N streptococci for isolation and rapid characterization of plasmid deoxyribonucleic acid. 总被引:30,自引:18,他引:12 下载免费PDF全文
Procedures for effective cellular lysis and plasmid deoxyribonucleic acid (DNA) isolation from group N streptococci were developed. Cells were grown at 32 degrees C for 4 h in a modified Elliker broth containing 20 mM DL-threonine. After cellular digestion with 2 mg of lysozyme per ml for 7 min at 37 degrees C, 1% sodium dodecyl sulfate exposure resulted in complete and immediate lysis. Lactose (Lac) plasmid species in Streptococcus lactis C2 and S. cremoris B1 (30 and 37 megadaltons, respectively) were demonstrated upon examination of DNA from the cleared lysates by agarose gel electrophoresis. Increasing the lysozyme treatment to 20 min or more resulted in loss of the Lac plasmid, whereas other resident plasmids were unaffected and demonstrable in agarose gels. Diethylpyrocarbonate added before lysis prevented Lac plasmid loss in 20-min lysozyme-treated cells, but was not effective after 40 min of lysozyme treatment. The results suggested that endogenous nuclease activity during the lysozyme treatment period initiated Lac plasmid DNA loss. The development of an efficient lysis procedure for the group N streptococci allowed rapid identification and characterization of plasmid DNA by agarose gel electrophoresis. The plasmid composition of S. lactis C2 and S. cremoris B1, as determined by agarose gel electrophoresis, compared favorably to previous electron microscopic observations. 相似文献
16.
THP基因的重新克隆及草菇表达载体的构建 总被引:3,自引:0,他引:3
草菇 (Volvariellavolvacea)是一种高温型的食用菌 ,在 4℃低温条件下 ,其菌丝自溶死亡 ,子实体发软、液化直至腐烂[1~ 3 ] 。草菇的这一特性严重地限制了草菇的生产、新鲜草菇的流通、低温冷冻保鲜和出口创汇以及草菇菌种的低温冷冻储藏。草菇是同宗结合的真菌 ,生活史复杂[4 ] ,菌丝没有锁状联合 ,杂种选择缺乏标记 ,这给草菇的杂交育种带来极大的困难[5,6] 。基因工程的发展为解决草菇不耐低温冷藏这一难题提供了可能。THP(ThermalHysteresisProtein)基因—热滞后蛋白基因 ,是加拿大科学… 相似文献
17.
目的:构建高效抑制核磷蛋白NPMl基因的短发夹RNA(shRNA)干扰载体。方法:以人NPM1基因为靶序列,设计并合成shRNA序列,将其连入RNA干扰慢病毒载体p113.7;酶切鉴定插入shRNA序列片段的质粒,经测序正确后转染293T细胞;Western印迹检测得到抑制效果好的载体pll-shRNA,将其-9慢病毒载体共转染293T细胞,进行病毒的包装,将得到的病毒感染HTl080细胞,通过RT-PCR、Western印迹等方法验证其抑制效果。结果:酶切证实构建的载体pll-shRNA中已插入外源基因片段,转染293T细胞后都有抑制效果,其中pll-shRNA2的抑制效果最好;用pll-shRNA2病毒感染HTl080细胞,RT-PCR和Western印迹检测分别在RNA和蛋白质水平证实NPMl的表达显著降低。结论:构建的RNA干扰载体pll-shRNA2能有效抑制NPMl的表达,为NPMl功能的研究提供了有力工具。 相似文献
18.
V M Andrianov N Iu Zemlianaia M Karimov M B Amerkhanova Iu Ts Vinetski? 《Genetika》1978,14(9):1503-1512
Fragments produced by digestion of Pisum sativum chloroplast DNA with EcoRI were examined by agarose gel electrophoresis. These EcoRI-fragments were joined in vitro to Apr-ColE1 RSF2124 plasmid and cloned in Escherichia coli. Methods of molecular cloning of plasmid chimeras by success gradient centrifugation and repeated transformation and selection of recombinant plasmids using mytomicin C were used for cloning hybrid plasmids with various EcoRI fragments of pea chloroplast DNA has been obtained. 相似文献
19.
20.
Plasmid DNA of molecular weight 6.8 × 106 was isolated from Streptomyces kasugaensis MB273. The plasmid DNA showed a single CsCl-ethidium bromide density gradient centrifugation, in neutral sucrose gradient centrifugation, and in agarose gel electrophoresis. When this DNA was digested with BamHI or SalI endonucleases, an unexpected number of fragments were found on agarose gel electrophoresis. Molecular weight summation of fragments obtained from double restriction enzyme digestions suggested that the plasmid DNA was a mixture of two different plasmids. This was confirmed by constructing recombinant plasmids between S. kasugaensis plasmid DNA and pBR322, and then by isolating two plasmids after SalI endonuclease treatment followed by sucrose gradient centrifugation. One of the plasmids (pSK1) had a single recognition site for BamHI, EcoRI, and SalI, and three sites for BglII. The other plasmid (pSK2) had a single recognition site for EcoRI and BglII, two recognition sites for BamHI, and no cleavage site for SalI. The cleavage maps of these plasmids were constructed using several restriction endonucleases. 相似文献