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为构建猪链球菌(Streptococcus suis)蛋白表面展示系统,本研究通过序列分析,确定猪链球菌的LPxTG蛋白及其信号肽(SP)和胞壁锚定基序(CWA),通过PCR扩增Peno-SP、GFP、CWA的DNA片段并融合,构建强启动子Peno控制表达编码SP-GFP-CWA融合蛋白的DNA片段,将该重组DNA片段连接pSET2载体,获得蛋白表面展示质粒,转化猪链球菌,构建得到以GFP为报告蛋白的猪链球菌蛋白表面展示系统。结果显示,利用猪链球菌的10个LPxTG蛋白及其SP和CWA序列,构建了10个含有Peno-SP-GFP-CWA融合片段的重组pSET2表面蛋白展示质粒pSsPSD1至pSsPSD10,分别转化猪链球菌05ZYH33,PCR鉴定显示其中7个转化猪链球菌。采用western blot初步检测其展示蛋白,结果显示,7个转化阳性菌株均能有效表达GFP蛋白,以成熟GFP条带为指标,均表现出了一定的外源GFP表面展示水平,分别命名为SsPSD1、SsPSD2、SsPSD4、SsPSD7-SsPSD10,其中SsPSD1、SsPSD4、SsPSD8和SsPSD9表面展示水平相对较好,在猪链球菌表面展示外源蛋白方面具有很好的潜力。本研究首次尝试建立猪链球菌蛋白表面展示系统,为猪链球菌表面递呈外源蛋白或抗原提供了新的策略。 相似文献
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XU Wan-yun WANG Hui-min WANG Wen-lun HU Meng-wei YAN Guo LI Jian-hua GAO Jian-feng 《中国畜牧兽医》2016,43(3):568-576
The specific primers were designed according to Ovis aries DRA gene sequence deposited in GenBank and the multiple cloning site of the plasmid pYD1,which was a vector used for protein surface display on Saccharomyces cerevisiae.The gene encoding DRA was amplified by PCR using the genomic RNA of Ovis aries.The 762 bp fragment was cloned and released in GenBank and registration number was KR422362.The PCR product was inserted into the yeast surface display plasmid vector pYD1 by double enzyme digestion.It was indicated that DRA gene was successfully integrated into the genome.Dot mutation was made at both ends of exon 2 in DRA gene for making restriction enzyme cutting site and design the exon 2 specific primers according to mutated Ovis aries DRA gene sequence.Sequenced exon 2 amplification products based on DNA pooling of sheep large sample template was analyzed the polymorphic loci.The polymorphic exon 2 246 bp fragment was obtained by double enzyme digestion and connected to surface display restructuring mutation carriers pYD1-DRA by the same double enzyme digestion,and then we successfully constructed yeast surface display libraries.We transformed it into Saccharomyces cerevisiae EBY100 cell.Yeast monoclone was identified by PCR amplification and sequencing,and we confirmed that DRA gene had been integrated into Saccharomyces cerevisiae genome.After galactose induced,it was detected that DRA gene library had been successfully demonstrated on the yeast cell surface under the fluorescence microscope by immunofluorescence method. 相似文献
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Utilization of digital differential display to identify differentially expressed genes related to rumen development 下载免费PDF全文
Daichi Kato Yutaka Suzuki Satoshi Haga KyoungHa So Eri Yamauchi Miwa Nakano Hiroshi Ishizaki Kichoon Choi Kazuo Katoh Sang‐Gun Roh 《Animal Science Journal》2016,87(4):584-590
This study aimed to identify the genes associated with the development of the rumen epithelium by screening for candidate genes by digital differential display (DDD) in silico. Using DDD in NCBI's UniGene database, expressed sequence tag (EST)‐based gene expression profiles were analyzed in rumen, reticulum, omasum, abomasum and other tissues in cattle. One hundred and ten candidate genes with high expression in the rumen were derived from a library of all tissues. The expression levels of 11 genes in all candidate genes were analyzed in the rumen, reticulum, omasum and abomasum of nine Japanese Black male calves (5‐week‐old pre‐weaning: n = 3; 15‐week‐old weaned calves: n = 6). Among the 11 genes, only 3‐hydroxy‐3‐methylglutaryl‐CoA synthase 2 (HMGCS2), aldo‐keto reductase family 1, member C1‐like (AKR1C1), and fatty acid binding protein 3 (FABP3) showed significant changes in the levels of gene expression in the rumen between the pre‐ and post‐weaning of calves. These results indicate that DDD analysis in silico can be useful for screening candidate genes related to rumen development, and that the changes in expression levels of three genes in the rumen may have been caused by weaning, aging or both. © 2015 Japanese Society of Animal Science 相似文献
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In order to detect the molecular mechanism of heterosis in pigs, the mRNA differential display technique was performed to investigate the differences of gene expression in the backfat tissue from Meishan,Large White and Meishan×Large White cross pigs.Nine 3'-end anchored primers in combination with ten 5'-end arbitrary primers were used to perform the differential display PCR and nearly 3 000 reproducible bands were examined .Fifteen expressed sepuence tags that were differentially expressed were isolated and then isdentifide through semi-quantitative RT-PCR.BLAST analysis revealed that the fifteen expressed sequence tags (ESTs) were ntt homologous to any of the known porcine genes or ESTs. These novel ESTs were then submitted to GenBank. 相似文献
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小鼠单个冷冻胚胎银染mRNA差异显示方法的建立 总被引:7,自引:2,他引:7
为建立单个冷冻胚胎银染mRNA差异显示方法,用小鼠单个4C鲜胚和冻胚RT—PCR产物,验证了冻胚和鲜胚实验结果的一致性。并从小鼠4C和8C冻胚的银染聚丙烯酰胺凝胶中回收了8C期的特异条带,通过再扩增、同收、克隆及酶切鉴定,克隆到了小鼠8C期胚胎差异表达的小鼠RP23—20A6基因。结果表明,所研究建立的单个冻胚银染mRNA差异显示方法具有可行性和有效性,可以用于动物早期胚胎基因表达的研究。 相似文献
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对mRNA差异显示技术中引物设计、应用非放射性标记、优化PCR参数、假阳性鉴定的方法等方面进行了概述。 相似文献
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