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排序方式: 共有586条查询结果,搜索用时 31 毫秒
31.
目的研究双质粒HBV DNA疫苗工程菌的中试发酵工艺。方法首先通过计算质粒拷贝数,检测工程菌DH5α/pS2.S和DH5α/pFP在传代过程中的遗传稳定性,通过摇瓶培养确定培养基组成,再在30L发酵罐内,通过改变培养基成分、培养时间、补料方式,确定最佳发酵参数。并将确定的最佳发酵参数应用于50L发酵罐连续3批中试规模的发酵,同时考察在发酵培养过程中质粒稳定性和超螺旋质粒DNA的比例。结果工程菌DH5α/pS2.S和DH5α/pFP在连续传代30次后,质粒拷贝数保持稳定。确定最佳发酵参数为以甘油为碳源的培养基培养,梯度恒速流加方式补料,培养时间为10h。通过3批稳定发酵,最终可获得湿菌58.0~71.8g/L,质粒含量可达到1.11~1.58mg/g菌,超螺旋质粒DNA的比例达93%以上。结论已建立了稳定的双质粒HBV DNA疫苗工程菌中试发酵工艺,为进一步规模化生产奠定了基础。 相似文献
32.
目的构建表达甲型H1N1流感病毒神经氨酸酶(Neuraminidase,NA)的重组腺病毒,并检测其免疫原性。方法从质粒pMD19T-simple-NA中扩增NA基因,克隆至穿梭质粒pShuttleCMV中,经同源重组获得重组腺病毒质粒,转染Ad-293细胞,包装出重组腺病毒Ad-NA,RT-PCR和免疫荧光法检测NA基因在Vero细胞中的转录和表达。CsCl密度梯度离心纯化重组腺病毒,免疫小鼠,ELISA法检测免疫小鼠血清中抗NA抗体滴度。结果重组腺病毒质粒经PacⅠ酶切鉴定表明带有目的基因的穿梭质粒已整合到腺病毒基因组中;NA基因在Vero细胞中成功转录和表达;重组腺病毒可刺激小鼠产生抗NA抗体,初免后4周,抗体水平达最高,为1∶100 000。结论成功构建了表达甲型H1N1流感病毒NA蛋白的重组腺病毒,其可刺激小鼠产生有效的免疫应答,为甲型H1N1流感病毒基因工程疫苗的研发奠定了基础。 相似文献
33.
34.
目的观察不同剂量重组弓形虫Peroxiredoxin蛋白(rTgPrx)诱导小鼠的体液免疫、细胞免疫应答及其抗弓形虫感染的保护作用。方法BALB/c小鼠60只,随机分为5组,分别用20、40、60、80μgrTgPrx(溶于100μlPBS中)和100μlPBS皮下免疫小鼠3次,间隔2周。末次免疫后第14天,用1×104个速殖子/只灌胃攻击,逐日观察小鼠健康状况。攻击后第30天,ELISA法检测小鼠血清IgG和小肠冲洗液sIgA水平,分离并计数脾淋巴细胞和小肠上皮内淋巴细胞(IEL),分离肝、脑组织内弓形虫速殖子并计数。结果60μgrTgPrx组小鼠血清IgG水平显著高于PBS、20μg和40μgrTgPrx组,小肠冲洗液sIgA组间比较差异无统计学意义;80μgrTgPrx组小鼠脾淋巴细胞数量明显高于PBS和20μgrTgPrx组,IEL数量各组间差异无统计学意义;60μg和80μgrTgPrx组小鼠脑和肝组织虫荷均低于PBS和20μgrTgPrx组。结论60μg和80μgrTgPrx皮下免疫小鼠可诱导有效的体液免疫、细胞免疫应答,且抗弓形虫感染的保护作用效果良好。 相似文献
35.
M Mete Altnta Betül Krdar Z lsen
nsan Kutlu
Ülgen 《Journal of chemical technology and biotechnology (Oxford, Oxfordshire : 1986)》2001,76(6):612-618
The recombinant Saccharomyces cerevisiae strain, YPB‐G, producing and secreting Bacillus subtilis α‐amylase and Aspergillus awamori glucoamylase as a fusion protein yielded efficient utilisation of starch. A segregated population balance model has been used to determine the probability of plasmid loss and plasmid copy number. The kinetics of cell growth and product (fusion protein) formation were based on a genetically structured model. The predictions were compared with the experimental observations obtained for the unstable recombinant S cerevisiae cells in a 1.5 dm?3 batch bioreactor with 30 g dm3 initial starch under non‐aerated conditions. The main advantage of the present model is that three different genetic classes were defined on the basis of the existence of plasmid and of the expression of the enzymes, ie cells containing plasmids and expressing the gene product, x1; cells containing plasmids and but not expressing the gene product, x2; and cells without plasmids, x3. It is confirmed by this model that the cells without plasmids outgrow and dominate in the fermentation medium (2.27 g dm?3 vs 0.51 g dm?3) as more and more glucose becomes available by the degradation of starch. © 2001 Society of Chemical Industry 相似文献
36.
Keita Aoki Reiko Nakajima Kanji Furuya Hironori Niki 《Yeast (Chichester, England)》2010,27(12):1049-1060
Schizosaccharomyces japonicus is a fission yeast for which new genetic tools have recently been developed. Here, we report novel plasmid vectors with high transformation efficiency and an electroporation method for Sz. japonicus. We isolated 44 replicating segments from 12 166 transformants of Sz. japonicus genomic fragments and found a chromosomal fragment, RS1, as a new replicating sequence that conferred high transformation activity to Sz. japonicus cells. This sequence was cloned into a pUC19 vector with ura4+ of Sz. pombe (pSJU11) or the kan gene on the kanMX6 module (pSJK11) as selection markers. These plasmids transformed Sz. japonicus cells in the early‐log phase by electroporation at a frequency of 123 cfu/µg for pSJK11 and 301 cfu/µg for pSJU11, which were higher than previously reported autonomously replicating sequences. Although a portion of plasmids remained in host cells by integration into the chromosome via RS1 segment, the plasmids could be recovered from transformants. The plasmid copy number was estimated to be 1.88 copies per cell by Southern blot analysis using a Sz. pombe ura4+ probe. The plasmid containing ade6+ suppressed the auxotrophic growth of the ade6‐domE mutant, indicating that the plasmid would be useful for suppressor screening and complementation assays in Sz. japonicus. Furthermore, pSJU11 transformed Sz. pombe cells with the same frequency as the pREP2 plasmid. This study is a report to demonstrate practical use of episomal plasmid vectors for genetic research in Sz. japonicus. RS1 has been submitted to the DDBJ/EMBL/GenBank database (Accession No. AB547343). Copyright © 2010 John Wiley & Sons, Ltd. 相似文献
37.
38.
Roy S. Tubb 《Journal of the Institute of Brewing》1987,93(2):91-96
Yeast genetics is now available as a practical tool for the development of brewing industry practices. The contribution of Brewing Research Foundation work (1978–84) to recent advances is illustrated by the construction of brewing strains with superattenuating (amylolytic) or anti-contaminant properties. Approaches based on hybridisation (by rare mating) or recombinant DNA technology have been evaluated. Techniques developed for (i) gene transfer to brewing strains, (ii) ensuring stable inheritance of novel characteristics and (iii) exploiting the secretory ability of yeast strains, can be widely applied not only with brewing, distilling, baking or wine yeasts, but also in the use of yeasts to produce novel biotechnical products. ‘Spin-off’ from these studies includes valuable methods for differentiating or enumerating wild yeasts in brewery quality control. 相似文献
39.
The predicted ORF3 polypeptide (Orf3p) of the linear genetic element pGKL2 from Kluyveromyces lactis was expressed in Bacillus megaterium as a fusion protein with a His(6X)-tag at the C-terminus for isolation by Ni-affinity chromatography. This is the first time that a yeast cytoplasmic gene product has been expressed heterologously as a functional protein in a bacterial system. The purified protein was found to display both RNA 5'-triphosphatase and guanylyltransferase activities. When the lysine residue present at position 177 of the protein within the sequence motif (KXDG), highly conserved in capping enzymes and other nucleotidyl transferases, was substituted by alanine, the guanylyltransferase activity was lost, thereby proving an important role for the transfer of GMP from GTP to the 5'-diphosphate end of the mRNA. Our in vitro data provides the first direct evidence that the polypeptide encoded by ORF3 of the cytoplasmic yeast plasmid pGKL2 functions as a plasmid-specific capping enzyme. Since genes equivalent to ORF3 of pGKL2 have been identified in all autonomous cytoplasmic yeast DNA elements investigated so far, our findings are of general significance for these widely distributed yeast extranuclear genetic elements. 相似文献
40.
Celia Conesa María Carmen Rota María-Dolores Pérez Miguel Calvo Lourdes Sánchez 《European Food Research and Technology》2008,228(2):205-211
The antibacterial activity of human lactoferrin from milk (hLF), recombinant human lactoferrin from Aspergillus awamori (rhLF) and their hydrolysates obtained with pepsin was investigated against Escherichia coli O157:H7, Salmonella Enteritidis and Listeria monocytogenes. The minimum inhibitory concentrations (MIC) and the minimum bactericidal concentrations (MBC) were determined for all the
bacteria and the proteins assayed. Taking into account the MICs found for both lactoferrins studied, we can say that they
behave very similarly, except for L. monocytogenes for which rhLF was more active. We studied the effect that heat treatments exerted on the antibacterial activity of the two
types of lactoferrin and the only heat treatment that had a negative effect on that activity was 85 °C for 10 min. The activity
of hLF and rhLF in UHT milk and whey against E. coli O157:H7 and L. monocytogenes, was also assayed. Our results showed a reduction in the number of viable cells for both microorganisms when were incubated
with rhLF or hLF, but this decrease was lower than in broth media. 相似文献