慢病毒载体介导的RNAi抑制牛整联蛋白亚基αv基因表达的研究
摘要
受体在病毒与宿主细胞的相互作用过程中起着极为重要的作用。口蹄疫病毒感染宿主细胞的第一步是病毒识别并结合存在于宿主细胞表面的受体,然后在受体的介导下,病毒才能够进入宿主细胞并在其中完成核酸的复制及病毒粒子的装配。整联蛋白是一类由α亚基和β亚基以非共价键结合形成的异源二聚体跨膜糖蛋白,能介导细胞与细胞、细胞与细胞外基质之间的相互作用,在细胞的增殖、分化、移行、生长等许多方面发挥重要作用。该蛋白家族拥有24个蛋白成员,其中,ανβ1、ανβ3、ανβ6、ανβ8是目前公认的FMDV的受体,这四种整联蛋白拥有一个共同的亚基αν。RNAi是由双链RNA介导的序列特异性RNA降解作用。它通过双链RNA的介导,特异性阻抑相关基因的表达,从而导致转录后水平的基因沉默。慢病毒载体是理想的真核细胞基因转移工具,被广泛应用于相关的RNAi研究领域,例如抗病毒研究、遗传性疾病的治疗、基因治疗。利用慢病毒载体稳定转染并整合宿主基因组的特性可以使设计好的RNA干扰片段在目的细胞系中持续表达,达到对靶基因持续干扰的效果。
本实验根据本课题组以前克隆的牛源整联蛋白αv基因(Genban登录号:DQ871215),按照siRNA设计原则,运用在线siRNA设计软件设计3个针对integrin-αv的siRNA靶点序列αν-2270、αν-1716、αν-483和一个siRNA阴性对照si0。运用实时荧光定量PCR技术筛选出高效的干扰片段αν-483,将αν-483的核苷酸序列交由生物公司合成其对应的双链cDNA,双链cDNA通过定向克隆连接至U6进入载体(U6 entry vector),构建出进入载体U6-αν483,U6-αν483与pLenti6/BLOCK-iT~(TM)载体系统中的表达质粒通过LR同源重组反应形成表达载体DEST-αν483。在生长状态良好的20代次之内的293FT细胞中,转染DEST-αν483及包装Mix中的三个包装质粒pLP1,pLP2和pLP/VSVG,72h之后收集慢病毒液,命名为plenti6/αv483。用pk-15进行慢病毒滴度的测定,结果显示plenti6/αv483的滴度为14.2×10~(-6) TU/ml,达到了转染细胞所需的滴度要求。用plenti6/αv483来转染牛肾细胞MDBK,在杀稻瘟菌素的抗性压力之下筛选出了四株单克隆的阳性细胞系,分别为1B8、1F5、2B9、2E6。经核酸水平的实时荧光定量PCR和细胞水平的cell-ELISA及间接免疫荧光实验鉴定,四株细胞系中整联蛋白的表达量均有不同程度的下降,其中1F5细胞株中整联蛋白表达量下调的幅度最大。
本研究筛选了具有高效干扰牛整联蛋白αv亚基基因的siRNA,以此为依据构建了表达具有干扰效果shRNA的慢病毒载体,并运用构建好的慢病毒载体筛选出了整联蛋白表达量下降的稳定转染细胞系。对通过下调病毒受体的表达来降低宿主对病毒敏感性的研究进行了初步探索。
Viral receptor plays a significant role in the interaction between virus and its cellular receptors. The attachment of foot-and-mouth-disease virus to the receptor on the surface of host cells is the first stage of virus infection, through which virus can entry the cell. Then virus completes the replication of its genome and the assembly of the viral particles. Integrins are a large family of heterodimeric transmembrane glycoproteins composed ofαandβsubmint that interact noncovalently at th cell surface. They mediate cell-cell interaction and the binding of cells to the extracellular matrix,and they play a crucial role in cell division, differentiation, migration, and survival. The protein family has 24 members. But onlyανβ1、ανβ3、ανβ6、ανβ8 four members have been identified as receptors for foot-and-mouth disease virus at present, and they have the same subunitαν. RNA interference refers to the inhibition of gene expression by dsRNA. It is a post transcriptional gene silencing mechanism, by which double stranded RNA specifically suppresses the expression of coherent sequences. As a good eukaryotic cells gene transfer vector, lentivirus-based vectors are generally applied in RNAi related research, such as antiviral research, therapy of hereditary diseases and gene therapy. Considering lentiviral vector can stably transfect host cells and even integrate into the host genomes, it can be used to express the designed siRNA in host cell. Thus continuously downregulating the express of the target gene.
According to the sequence of bovine integrin published in Genbank, we designed three siRNAs targeting integrinανsubunit by the online software of designing siRNA namedαν-2270、αν-1716、αν-483. Moreover, a negative control named si0 is also made. The most efficient siRNA(αν-483) was selected by real-time RT-PCR. Authorized a biotechnology company to synthetize the corresponding ds oligo cDNA sequence ofαν-483 and the cloned the ds oligo into the U6 entry vector, thus construst the entry vector U6-αν483. After the LR recombination reaction between the entry vector U6-αν483 and the expression vector in the pLenti6/BLOCK-iT~(TM) system, the sequence of the U6 RNAi cassette in U6-αν483 has been transferred into the destination vector and the expression vector named DEST-αν483 was constructed. Transfected the DEST-αν483 and the three packaging plasmids pLP1,pLP2 and pLP/VSVG in the packaging mix into well-grown 293FT and harvested virus(named plenti6/αv483)-containing supernatants 72 hours posttransfection. After Titering the plenti6/αv483 lentiviral stock using pk-15 cell line, we figured the titer of the lentiviral construct out: 14.2×10~(-6) TU/ml. Then MDBK cells were infected by plenti6/αv483 and were continually seeded in the medium at the optimal blasticidin regulatory concentration for two weeks to obtain the blasticidin resistant colonies. At last, four resistant cells lines named 1B8、1F5、2B9、2E6 were selected and then cultured for positive identification. After the identification by real-time RT-PCR in the nucleic acid level and the cell-ELISA、IIF in the protein level, we confirmed that the expression of integrin in these four cells lines all had different degree of decline. Furthermore, the downregulating expression of integrin in 1F5 cell line is the most significant.
The experiment had constructed the lentiviral vector which express the shRNA targeting the bovine integrinανsubunit successfully and then obtained blasticidin resistant cells whose genome had been integrated by the lentiviral vector. We had carried out the preliminary study of anti-virus by downregulating the expression of the viral receptor.
本实验根据本课题组以前克隆的牛源整联蛋白αv基因(Genban登录号:DQ871215),按照siRNA设计原则,运用在线siRNA设计软件设计3个针对integrin-αv的siRNA靶点序列αν-2270、αν-1716、αν-483和一个siRNA阴性对照si0。运用实时荧光定量PCR技术筛选出高效的干扰片段αν-483,将αν-483的核苷酸序列交由生物公司合成其对应的双链cDNA,双链cDNA通过定向克隆连接至U6进入载体(U6 entry vector),构建出进入载体U6-αν483,U6-αν483与pLenti6/BLOCK-iT~(TM)载体系统中的表达质粒通过LR同源重组反应形成表达载体DEST-αν483。在生长状态良好的20代次之内的293FT细胞中,转染DEST-αν483及包装Mix中的三个包装质粒pLP1,pLP2和pLP/VSVG,72h之后收集慢病毒液,命名为plenti6/αv483。用pk-15进行慢病毒滴度的测定,结果显示plenti6/αv483的滴度为14.2×10~(-6) TU/ml,达到了转染细胞所需的滴度要求。用plenti6/αv483来转染牛肾细胞MDBK,在杀稻瘟菌素的抗性压力之下筛选出了四株单克隆的阳性细胞系,分别为1B8、1F5、2B9、2E6。经核酸水平的实时荧光定量PCR和细胞水平的cell-ELISA及间接免疫荧光实验鉴定,四株细胞系中整联蛋白的表达量均有不同程度的下降,其中1F5细胞株中整联蛋白表达量下调的幅度最大。
本研究筛选了具有高效干扰牛整联蛋白αv亚基基因的siRNA,以此为依据构建了表达具有干扰效果shRNA的慢病毒载体,并运用构建好的慢病毒载体筛选出了整联蛋白表达量下降的稳定转染细胞系。对通过下调病毒受体的表达来降低宿主对病毒敏感性的研究进行了初步探索。
Viral receptor plays a significant role in the interaction between virus and its cellular receptors. The attachment of foot-and-mouth-disease virus to the receptor on the surface of host cells is the first stage of virus infection, through which virus can entry the cell. Then virus completes the replication of its genome and the assembly of the viral particles. Integrins are a large family of heterodimeric transmembrane glycoproteins composed ofαandβsubmint that interact noncovalently at th cell surface. They mediate cell-cell interaction and the binding of cells to the extracellular matrix,and they play a crucial role in cell division, differentiation, migration, and survival. The protein family has 24 members. But onlyανβ1、ανβ3、ανβ6、ανβ8 four members have been identified as receptors for foot-and-mouth disease virus at present, and they have the same subunitαν. RNA interference refers to the inhibition of gene expression by dsRNA. It is a post transcriptional gene silencing mechanism, by which double stranded RNA specifically suppresses the expression of coherent sequences. As a good eukaryotic cells gene transfer vector, lentivirus-based vectors are generally applied in RNAi related research, such as antiviral research, therapy of hereditary diseases and gene therapy. Considering lentiviral vector can stably transfect host cells and even integrate into the host genomes, it can be used to express the designed siRNA in host cell. Thus continuously downregulating the express of the target gene.
According to the sequence of bovine integrin published in Genbank, we designed three siRNAs targeting integrinανsubunit by the online software of designing siRNA namedαν-2270、αν-1716、αν-483. Moreover, a negative control named si0 is also made. The most efficient siRNA(αν-483) was selected by real-time RT-PCR. Authorized a biotechnology company to synthetize the corresponding ds oligo cDNA sequence ofαν-483 and the cloned the ds oligo into the U6 entry vector, thus construst the entry vector U6-αν483. After the LR recombination reaction between the entry vector U6-αν483 and the expression vector in the pLenti6/BLOCK-iT~(TM) system, the sequence of the U6 RNAi cassette in U6-αν483 has been transferred into the destination vector and the expression vector named DEST-αν483 was constructed. Transfected the DEST-αν483 and the three packaging plasmids pLP1,pLP2 and pLP/VSVG in the packaging mix into well-grown 293FT and harvested virus(named plenti6/αv483)-containing supernatants 72 hours posttransfection. After Titering the plenti6/αv483 lentiviral stock using pk-15 cell line, we figured the titer of the lentiviral construct out: 14.2×10~(-6) TU/ml. Then MDBK cells were infected by plenti6/αv483 and were continually seeded in the medium at the optimal blasticidin regulatory concentration for two weeks to obtain the blasticidin resistant colonies. At last, four resistant cells lines named 1B8、1F5、2B9、2E6 were selected and then cultured for positive identification. After the identification by real-time RT-PCR in the nucleic acid level and the cell-ELISA、IIF in the protein level, we confirmed that the expression of integrin in these four cells lines all had different degree of decline. Furthermore, the downregulating expression of integrin in 1F5 cell line is the most significant.
The experiment had constructed the lentiviral vector which express the shRNA targeting the bovine integrinανsubunit successfully and then obtained blasticidin resistant cells whose genome had been integrated by the lentiviral vector. We had carried out the preliminary study of anti-virus by downregulating the expression of the viral receptor.
引文
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