目的:检测Ras相关区域家族1A(Ras association domain family 1A,RASSF1A)在胰腺癌细胞株中的甲基化和表达状态,探讨其启动子异常甲基化在胰腺癌发病过程中的作用。方法:采用重亚硫酸盐测序PCR(bisulfite genomic sequencing PCR,BSP)联...目的:检测Ras相关区域家族1A(Ras association domain family 1A,RASSF1A)在胰腺癌细胞株中的甲基化和表达状态,探讨其启动子异常甲基化在胰腺癌发病过程中的作用。方法:采用重亚硫酸盐测序PCR(bisulfite genomic sequencing PCR,BSP)联合TA克隆测序检测胰腺癌细胞株PANC-1及胰腺癌组织、癌旁组织及正常胰腺组织中RASSF1A启动子区CpG岛的甲基化状态,以甲基化酶抑制剂5-aza-2-deoxycitydine(5-aza-dC)处理PANC-1,观察处理前后甲基化率变化情况,逆转录PCR观察RASSF1A的mRNA表达情况。结果:在PANC-1细胞中RASSF1A启动子的甲基化率平均为100.00%,在正常胰腺、癌旁及癌组织中平均分别为1.79%、93.75%和100.00%,与正常胰腺组织相比,胰腺癌旁及癌组织的RASSF1A启动子甲基化率明显增高(P<0.01),而癌旁及癌组织之间无明显差异(P>0.05)。在PANC-1细胞、胰腺癌组织及癌旁组织中RASSF1A基因无表达,在正常胰腺组织中RASSF1A基因呈阳性表达;PANC-1细胞经5-aza-dC处理后,RASSF1A的甲基化率下降(88.89%,P<0.05),mRNA表达无变化。结论:胰腺癌细胞株PANC-1及癌组织、癌旁组织RASSF1A基因表达与启动子区甲基化状态有关,启动子区的高甲基化导致PANC-1中RASSF1A基因的表达沉默。该基因异常甲基化有望成为胰腺癌的早期诊断指标和治疗靶点。展开更多
在基因克隆操作中,由于常用克隆载体多克隆位点的酶切位点数目有限,造成一些DNA片段的克隆、连接受限。为解决这一问题,本文利用pEASY-T1 S imp le载体无酶切位点的特点,通过TA克隆的方法引入多个单一酶切位点,并对所构建的克隆载体进...在基因克隆操作中,由于常用克隆载体多克隆位点的酶切位点数目有限,造成一些DNA片段的克隆、连接受限。为解决这一问题,本文利用pEASY-T1 S imp le载体无酶切位点的特点,通过TA克隆的方法引入多个单一酶切位点,并对所构建的克隆载体进行了验证。最后证明,这种方法可高效构建含有不同多克隆位点的载体,克服了对常用克隆载体的依赖,大大提高了基因操作的灵活性和有效性。展开更多
The Bacillus strain BH072 isolated from a honey sample showed strong antifungal activity against phytopathogen. Gene cloning test demonstrated that the strain had a tas A gene encoding an antifungal Tas A protein. Alt...The Bacillus strain BH072 isolated from a honey sample showed strong antifungal activity against phytopathogen. Gene cloning test demonstrated that the strain had a tas A gene encoding an antifungal Tas A protein. Although the wild strain simultaneously produced various antifungal substances, only the physicochemical property and antifungal activity of Tas A protein were unclear due to the difficulty in extraction. In this study, tas A gene encoding the protein from Bacillus sp. BH072 was amplified by using the polymerase chain reaction(PCR) method and cloned into p ET 28a(+) vector, and then expressed in host cells Escherichia coli BL21(DE3). The expressed proteins were collected by centrifugation and ultrasonic treatment, and then purified by using nickel-nitrilotriacetic acid(Ni-NTA)metal affinity column and dialysis methods. The result of sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) test showed that an expected protein band appeared with a size of 31 k Da. The expressed products possessed antifungal activity against the phytopathogenic indicator strain Botrytis cinerea. A genetically engineered strain tas A of E. coli was established in this study which can efficiently express Tas A protein.展开更多
文摘目的:检测Ras相关区域家族1A(Ras association domain family 1A,RASSF1A)在胰腺癌细胞株中的甲基化和表达状态,探讨其启动子异常甲基化在胰腺癌发病过程中的作用。方法:采用重亚硫酸盐测序PCR(bisulfite genomic sequencing PCR,BSP)联合TA克隆测序检测胰腺癌细胞株PANC-1及胰腺癌组织、癌旁组织及正常胰腺组织中RASSF1A启动子区CpG岛的甲基化状态,以甲基化酶抑制剂5-aza-2-deoxycitydine(5-aza-dC)处理PANC-1,观察处理前后甲基化率变化情况,逆转录PCR观察RASSF1A的mRNA表达情况。结果:在PANC-1细胞中RASSF1A启动子的甲基化率平均为100.00%,在正常胰腺、癌旁及癌组织中平均分别为1.79%、93.75%和100.00%,与正常胰腺组织相比,胰腺癌旁及癌组织的RASSF1A启动子甲基化率明显增高(P<0.01),而癌旁及癌组织之间无明显差异(P>0.05)。在PANC-1细胞、胰腺癌组织及癌旁组织中RASSF1A基因无表达,在正常胰腺组织中RASSF1A基因呈阳性表达;PANC-1细胞经5-aza-dC处理后,RASSF1A的甲基化率下降(88.89%,P<0.05),mRNA表达无变化。结论:胰腺癌细胞株PANC-1及癌组织、癌旁组织RASSF1A基因表达与启动子区甲基化状态有关,启动子区的高甲基化导致PANC-1中RASSF1A基因的表达沉默。该基因异常甲基化有望成为胰腺癌的早期诊断指标和治疗靶点。
文摘在基因克隆操作中,由于常用克隆载体多克隆位点的酶切位点数目有限,造成一些DNA片段的克隆、连接受限。为解决这一问题,本文利用pEASY-T1 S imp le载体无酶切位点的特点,通过TA克隆的方法引入多个单一酶切位点,并对所构建的克隆载体进行了验证。最后证明,这种方法可高效构建含有不同多克隆位点的载体,克服了对常用克隆载体的依赖,大大提高了基因操作的灵活性和有效性。
文摘The Bacillus strain BH072 isolated from a honey sample showed strong antifungal activity against phytopathogen. Gene cloning test demonstrated that the strain had a tas A gene encoding an antifungal Tas A protein. Although the wild strain simultaneously produced various antifungal substances, only the physicochemical property and antifungal activity of Tas A protein were unclear due to the difficulty in extraction. In this study, tas A gene encoding the protein from Bacillus sp. BH072 was amplified by using the polymerase chain reaction(PCR) method and cloned into p ET 28a(+) vector, and then expressed in host cells Escherichia coli BL21(DE3). The expressed proteins were collected by centrifugation and ultrasonic treatment, and then purified by using nickel-nitrilotriacetic acid(Ni-NTA)metal affinity column and dialysis methods. The result of sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) test showed that an expected protein band appeared with a size of 31 k Da. The expressed products possessed antifungal activity against the phytopathogenic indicator strain Botrytis cinerea. A genetically engineered strain tas A of E. coli was established in this study which can efficiently express Tas A protein.