通过EST和锚定PCR技术,从海湾扇贝(Argopecten irradians)中克隆得到一种溶菌酶基因的全长cDNA(全长659bp,编码200个氨基酸).BLASTx分析的结果显示,它和脊椎动物g型溶菌酶相似性较高,却不同于无脊椎动物中已发现的c型和i型溶菌酶.在其...通过EST和锚定PCR技术,从海湾扇贝(Argopecten irradians)中克隆得到一种溶菌酶基因的全长cDNA(全长659bp,编码200个氨基酸).BLASTx分析的结果显示,它和脊椎动物g型溶菌酶相似性较高,却不同于无脊椎动物中已发现的c型和i型溶菌酶.在其编码的氨基酸序列中发现了g型溶菌酶的活性中心(Glu 82, Asp 97, Asp 108),同时6个保守的半胱氨酸也与鸟类和鱼类的g型溶菌酶相一致.结合BLASTX分析的结果,可以确认所获得的cDNA序列是一种在无脊椎动物中首次发现的g型溶菌酶的编码序列.采用Clustalw 软件,对多种脊椎动物c型、g型溶菌酶和无脊椎动物c型、i型溶菌酶以及本研究发现的无脊椎动物g型溶菌酶进行了分析,结果发现,无脊椎动物c型、i型溶菌酶和脊椎动物c型溶菌酶同源性较高,而海湾扇贝g型溶菌酶和脊椎动物g型溶菌酶同源性较高.由此说明c型、g型溶菌酶从无脊椎到脊椎动物的进化是平行发生的,这对进一步研究溶菌酶及其分子进化具有重要意义.展开更多
AIM: To develop and optimize cDNA representational difference analysis (cDNA RDA) method and to identify and clone garlic up-regulated genes in human gastric cancer (HGC) cells. METHODS: We performed cDNA RDA method b...AIM: To develop and optimize cDNA representational difference analysis (cDNA RDA) method and to identify and clone garlic up-regulated genes in human gastric cancer (HGC) cells. METHODS: We performed cDNA RDA method by using abundant double-stranded cDNA messages provided by two self-constructed cDNA libraries (Allitridi-treated and paternal HGC cell line BGC823 cells cDNA libraries respectively). Bam H I and Xho I restriction sites harbored in the library vector were used to select representations. Northern and Slot blots analyses were employed to identify the obtained difference products. RESULTS: Fragments released from the cDNA library vector after restriction endonuclease digestion acted as good marker indicating the appropriate digestion degree for library DNA. Two novel expressed sequence tags (ESTs) and a recombinant gene were obtained. Slot blots result showed a 8-fold increase of glia-derived nexin/protease nexin 1 (GDN/PN1) gene expression level and 4-fold increase of hepatitis B virus x-interacting protein (XIP) mRNA level in BGC823 cells after Allitridi treatment for 72h. CONCLUSION: Elevated levels of GDN/PN1 and XIP mRNAs induced by Allitridi provide valuable molecular evidence for elucidating the garlic's efficacies against neurodegenerative and inflammatory diseases. Isolation of a recombinant gene and two novel ESTs further show cDNA RDA based on cDNA libraries to be a powerful method with high specificity and reproducibility in cloning differentially expressed genes.展开更多
文摘以经产荷斯坦奶牛外周血白细胞为材料,分离Poly(A)+ RNA,反转录合成单链及双链cDNA,经酶切成平均大小为400～600 bp的片段,将患乳房炎奶牛cDNA分成2组,分别与2种不同的接头连接,再与健康奶牛cDNA进行2次消减杂交和2次抑制性PCR扩增,将第2次PCR产物与pGEM-T载体连接,转化大肠杆菌TOP10感受态细胞进行文库扩增,构建了具有高消减效率的奶牛乳房炎抗性相关cDNA文库.文库扩增后得到610个白色阳性克隆,随机挑选克隆进行PCR鉴定,插入片段主要分布在250～750 bp 之间.文库的成功构建为进一步筛选、克隆与奶牛乳房炎抗性相关的基因奠定了基础,对研究奶牛乳房炎抗性的分子机制以及乳房炎的综合防制具有重要意义.
文摘通过EST和锚定PCR技术,从海湾扇贝(Argopecten irradians)中克隆得到一种溶菌酶基因的全长cDNA(全长659bp,编码200个氨基酸).BLASTx分析的结果显示,它和脊椎动物g型溶菌酶相似性较高,却不同于无脊椎动物中已发现的c型和i型溶菌酶.在其编码的氨基酸序列中发现了g型溶菌酶的活性中心(Glu 82, Asp 97, Asp 108),同时6个保守的半胱氨酸也与鸟类和鱼类的g型溶菌酶相一致.结合BLASTX分析的结果,可以确认所获得的cDNA序列是一种在无脊椎动物中首次发现的g型溶菌酶的编码序列.采用Clustalw 软件,对多种脊椎动物c型、g型溶菌酶和无脊椎动物c型、i型溶菌酶以及本研究发现的无脊椎动物g型溶菌酶进行了分析,结果发现,无脊椎动物c型、i型溶菌酶和脊椎动物c型溶菌酶同源性较高,而海湾扇贝g型溶菌酶和脊椎动物g型溶菌酶同源性较高.由此说明c型、g型溶菌酶从无脊椎到脊椎动物的进化是平行发生的,这对进一步研究溶菌酶及其分子进化具有重要意义.
基金the Natural Scientific Foundation of China (NSFC3962526)National High-Technology Project-863 (102-10-01-04)
文摘AIM: To develop and optimize cDNA representational difference analysis (cDNA RDA) method and to identify and clone garlic up-regulated genes in human gastric cancer (HGC) cells. METHODS: We performed cDNA RDA method by using abundant double-stranded cDNA messages provided by two self-constructed cDNA libraries (Allitridi-treated and paternal HGC cell line BGC823 cells cDNA libraries respectively). Bam H I and Xho I restriction sites harbored in the library vector were used to select representations. Northern and Slot blots analyses were employed to identify the obtained difference products. RESULTS: Fragments released from the cDNA library vector after restriction endonuclease digestion acted as good marker indicating the appropriate digestion degree for library DNA. Two novel expressed sequence tags (ESTs) and a recombinant gene were obtained. Slot blots result showed a 8-fold increase of glia-derived nexin/protease nexin 1 (GDN/PN1) gene expression level and 4-fold increase of hepatitis B virus x-interacting protein (XIP) mRNA level in BGC823 cells after Allitridi treatment for 72h. CONCLUSION: Elevated levels of GDN/PN1 and XIP mRNAs induced by Allitridi provide valuable molecular evidence for elucidating the garlic's efficacies against neurodegenerative and inflammatory diseases. Isolation of a recombinant gene and two novel ESTs further show cDNA RDA based on cDNA libraries to be a powerful method with high specificity and reproducibility in cloning differentially expressed genes.
