猪MyD88基因干扰重组慢病毒载体的构建、病毒包装及效果评价

doi:10.11843/j.issn.0366-6964.2015.04.021

Abstract

Abstract:

Myeloid differentiation factor 88 (MyD88)，an important adaptor protein in TLRs/IL-1R signaling pathways，plays an important role in immune response and disease prevention.This study intends to obtain pig small intestinal epithelial cells in which MyD88 gene is silent mediated by lentiviru，as an effective model for analyzing molecular mechanism of intestinal disease caused by pathogenic bacteria.In this study，four plasmid expression vectors were constructed，which coded shRNAs against pig MyD88 gene，and the over-expression vector coding MyD88 gene was also constructed.By the method of real-time fluorescent quantitative PCR，the level of MyD88 gene in the cotransfected 293 cells was detected.The highest efficiency vector was adopted and packaged into lentiviral vector to obtain pig small intestinal epithelial cells with silent MyD88 gene.The IPEC-J2 cell was obtained in which the MyD88 mRNA expression was reduced by 69.3%，meeting the requirement for gene function analysis.The obtained pig intestinal epithelial cell with stable MyD88 gene silencing provides important material for mechanism research of TLRs/IL-1R signal pathway caused by pathogenic microorganisms in pig intestines.

CLC Number:

S852.2

ZI Chen，XIA Ri-wei，YIN Xue-mei，YU Li-huai，ZHU Guo-qiang，WU Sheng-long，BAO Wen-bin. Construction and Packaging of Recombinant Lentivirus Interference Vector Specific to Porcine MyD88 Gene and the Evaluation of the Corresponding Lentivirus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(4): 657-664.

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References

［1］MUZIO M，POLENTARUTTI N，BOSISIO D，et al.Toll-like receptor family and signalling pathway［J］.Biochem Soc Trans，2000，28(5)：563-566.
［2］GHOSH S，KARIN M.Missing pieces in the NF-kappaB puzzle［J］.Cell，2002，109(Suppl)：S81-S96.
［3］JANSSENS S，BEYAERT R.Functional diversity and regulation of different interleukin-1 receptor-associated kinase (IRAK) family members［J］.Mol Cell，2003，11(2)：293-302.
［4］O′NEILL L A.Signal transduction pathways activated by the IL-1 receptor/toll-like receptor superfamily［J］.Curr Top Microbiol Immunol，2002，270：47-61.
［5］SUZUKI N，SUZUKI S，YEH W C.IRAK-4 as the central TIR signaling mediator in innate immunity［J］.Trends Immunol，2002，23(10)：503-506.
［6］SUZUKI N，SUZUKI S，DUNCAN G S，et al.Severe impairment of interleukin-1 and Toll-like receptor signalling in mice lacking IRAK-4［J］. Nature，2002，416(6882)：750-756.
［7］LI X，LIU H，YANG S，et al.Characterization analysis and polymorphism detection of the porcine MyD88 gene［J］.Genet Mol Biol，2009，32(2)：295-300.
［8］TOHNO M，SHIMAZU T，ASO H，et al.Molecular cloning and functional characterization of porcine MyD88 essential for TLR signaling［J］.Cell Mol Immunol，2007，4(5)：369-376.
［9］MOSES T，WAGNER L，FLEMING S D.TLR4-mediated Cox-2 expression increases intestinal ischemia/reperfusion-induced damage［J］. J Leukoc Biol，2009，86(4)：971-980.
［10］LOIARRO M，CAPOLUNGHI F，FANTÒ N，et al.Pivotal advance：Inhibition of MyD88 dimerization and recruitment of IRAK1 and IRAK4 by a novel peptidomimetic compound［J］.J Leukoc Biol，2007，82(4)：801-810.
［11］POLTORAK A，HE X，SMIRNOVA I，et al.Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice：mutations in Tlr4 gene［J］.Science，1998，282(5396)：2085-2088.
［12］KAWAI T，ADACHI O，OGAWA T，et al.Unresponsiveness of MyD88-deficient mice to endotoxin［J］.Immunity，1999，11(1)：115-122.
［13］TAUSZIG-DELAMASURE S，BILAK H，CAPOVILLA M，et al.Drosophila MyD88 is required for the response to fungal and Gram-positive bacterial infections［J］.Nat Immunol，2002，3(1)：91-97.
［14］YAMAWAKI Y，KIMURA H，HOSOI T，et al.MyD88 plays a key role in LPS-induced Stat3 activation in the hypothalamus［J］. Am J Physiol Regul Integr Comp Physiol，2010，298(2)：R403-410.
［15］DROUIN-OUELLET J，LEBEL M，FILALI M，et al.MyD88 deficiency results in both cognitive and motor impairments in mice［J］.Brain Behav Immun，2012，26(6)：880-885.
［16］罗庆苗，苗向阳，张瑞杰.转基因动物新技术研究进展［J］.遗传，2011，33(5)：449-458.
LUO Q M，MIAO X Y，ZHANG R J.An update on the development of transgenic animal technology［J］.Hereditas (Beijing)，2011，33(5)：449-458.（in Chinese）
［17］杨浩，李新建，程佳，等.慢病毒介导shRNA干扰颗粒体蛋白前体(GRN)促进猪前体脂肪细胞分化［J］.农业生物技术学报，2012，20(6)：599-605.
YANG H，LI X J，CHENG J，et al.Lentivirus mediated granulin shRNA interference silencing promotes pig preadipocyte differentiation［J］.Journal of Agricultural Biotechnology，2012，20(6)：599-605.（in Chinese）
［18］程佳，蒲蕾，杨浩，等.慢病毒介导shRNA干扰RBP4促进猪前体脂肪细胞成脂分化［J］.畜牧兽医学报，2013，44(6)：937-943.
CHENG J，PU L，YANG H，et al.Lentivirus mediated shRNA interference silencing RBP4 promoted differentiation of porcine preadipocytes［J］.Acta Veterinaria et Zootechnica Sinica，2013，44(6)：937-943.（in Chinese）
［19］LAN Y，ZHAO K，HE W，et al.Inhibition of porcine hemagglutinating encephalomyelitis virus replication by short hairpin RNAs targeting of the nucleocapsid gene in a porcine kidney cell line［J］.J Virol Methods，2012，179(2)：414-418.
［20］BAVYKIN A S，MISHIN D V，KARPUKHIN A V，et al.The ability of small interfering RNA oligonucleotides to decrease the infective activity of hepatitis C virus in the cell cultures［J］.Vopr Virusol，2012，57(2)：32-34.
［21］周谦君，范慧敏，刘中民.MyD88siRNA诱导猪调节性树突状细胞的实验研究［J］.中国细胞生物学学报，2010，32(3)：415-421.
ZHOU Q J，FAN H M，LIU Z M.MyD88 knockdown by liposome-mediated RNA interference to porcine monocyte-derived dendritic cell［J］.Chinese Journal of Cell Biology，2010，32(3)：415-421.（in Chinese）

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Construction and Packaging of Recombinant Lentivirus Interference Vector Specific to Porcine MyD88 Gene and the Evaluation of the Corresponding Lentivirus

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