鸡膜联蛋白A2在DF-1细胞中的表达及亚细胞定位
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摘要
根据GenBank已发表的鸡膜联蛋白A2(ANXA2)基因序列,设计合成一对特异性引物从DF-1细胞总RNA的反转录产物中扩增鸡ANXA2基因开放阅读框,通过酶切、连接等方法将其亚克隆到真核表达载体pEGFP-C1,构建重组真核表达载体pEGFP-ANXA2。利用脂质体转染法将pEGFP-ANXA2转染至DF-1细胞,24 h后分别检测融合蛋白EGFP-ANXA2在细胞中的表达及亚细胞定位情况。结果显示:成功构建了鸡ANXA2基因的重组真核表达载体pEGFP-ANXA2;将其转染DF-1细胞后得到与预期大小相符的EGFP-ANXA2蛋白条带,荧光显微镜观察显示EGFP-ANXA2主要定位在细胞质。研究结果为进一步开展鸡ANXA2在相关家禽病毒复制中的作用研究奠定了基础。
A pair of specific primers was designed to amplify the ORF region of chicken Annexin A2(ANXA2)gene from the reverse transcription products of the DF-1 total RNA. The obtained PCR products were then subcloned into the eukaryotic expression vector pEGFP-C1 to construct the recombinant plasmid pEGFP-ANXA2. The recombinant plasmid was transfected into DF-1 cells by liposome transfection,and the expression and subcellular localization of the fusion protein EGFP-ANXA2 were detected at 24 h. The results showed that the recombinant plasmid pEGFP-ANXA2 was successfully constructed and the expected fusion protein EGFP-ANXA2 was correctly expressed in DF-1 cells.Fluorescence analysis indicated that EGFP-ANXA2 mainly localized in the cytoplasm.These results will lay the foundation for further studying the role of chicken ANXA2 in the replication of the related avian viruses.
A pair of specific primers was designed to amplify the ORF region of chicken Annexin A2(ANXA2)gene from the reverse transcription products of the DF-1 total RNA. The obtained PCR products were then subcloned into the eukaryotic expression vector pEGFP-C1 to construct the recombinant plasmid pEGFP-ANXA2. The recombinant plasmid was transfected into DF-1 cells by liposome transfection,and the expression and subcellular localization of the fusion protein EGFP-ANXA2 were detected at 24 h. The results showed that the recombinant plasmid pEGFP-ANXA2 was successfully constructed and the expected fusion protein EGFP-ANXA2 was correctly expressed in DF-1 cells.Fluorescence analysis indicated that EGFP-ANXA2 mainly localized in the cytoplasm.These results will lay the foundation for further studying the role of chicken ANXA2 in the replication of the related avian viruses.
引文
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[8] MA Y, SUN J, GU L, et al. Annexin A2(ANXA2)interacts with nonstructural protein 1 and promotes the replication of highly pathogenic H5N1 avian influenza virus[J].BMC Microbiol, 2017, 17(1):191.
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[12] SHENG C, LIU X, JIANG Q, et al. Annexin A2 is involved in the production of classical swine fever virus infectious particles[J]. J Gen Virol, 2015, 96(5):1027-1032.
[13] ZHANG C, XUE C, LI Y, et al. Profiling of cellular proteins in porcine reproductive and respiratory syndrome virus virions by proteomics analysis[J]. Virol J, 2010, 7:242.
[14] RESCHER U, GERKE V. Annexins-unique membrane binding proteins with diverse functions[J]. J Cell Sci,2004, 117(13):2631-2639.
[15] BABIYCHUK E B, DRAEGER A. Regulation of ecto-5'-nucleotidase activity via Ca2+-dependent, annexin 2-mediated membrane rearrangement?[J]. Biochem Soc Trans,2006, 34(3):374-376.
[16] JOST M, GERKE V. Mapping of a regulatory important site for protein kinase C phosphorylation in the N-terminal domain of annexinⅡ[J]. Biochim Biophys Acta,1996, 1313(3):283-289.
[17] SHAO C, ZHANG F, KEMP M M, et al. Crystallographic analysis of calcium-dependent heparin binding to annexin A2[J]. J Biol Chem, 2006, 281(42):31689-31695.
[18] MA G, GREENWELL-WILD T, LEI K, et al. Secretory leukocyte protease inhibitor binds to annexinⅡ, a cofactor for macrophage HIV-1 infection[J]. J Exp Med,2004, 200(10):1337-1346.
[19] PELCHEN-MATTHEWS A, KRAMER B, MARSH M. Infectious HIV-1 assembles in late endosomes in primary macrophages[J]. J Cell Biol, 2003, 162(3):443-455.
[20] BEATON A R, RODRIGUEZ J, REDDY Y K, et al. The membrane trafficking protein calpactin forms a complex with bluetongue virus protein NS3 and mediates virus release[J]. Proc Natl Acad Sci, 2002, 99(20):13154-13159.
[21] YANG Z, SHI Z, GUO H, et al. Annexin 2 is a host protein binding to classical swine fever virus E2 glycoprotein and promoting viral growth in PK-15 cells[J]. Virus Res, 2015, 201:16-23.
[22] YANG S L, CHOU Y T, WU C N, et al. AnnexinⅡbinds to capsid protein VP1 of enterovirus 71 and enhances viral infectivity[J]. J Virol, 2011, 85(22):11809-11820.
[23] SU H, YANG X, WANG S, et al. Effect of annexinⅡ-mediated conversion of plasmin from plasminogen on airborne transmission of H9N2 avian influenza virus[J]. Vet Microbiol, 2018, 223:100-106.
[2] HAYES M J, SHAO D, BAILLY M, et al. Regulation of actin dynamics by annexin 2[J]. EMBO J, 2006, 25(9):1816-1826.
[3]陈江涛,吉艳红,王光祥,等.膜联蛋白A2参与病毒复制循环研究进展[J].中国人兽共患病学报,2010,26(6):592-595.
[4] FREED E O, MARTIN M A.HIVs and their replication[J].Fields Virology. Lippincott Williams&Wilkins, Philadelphia, PA. 2001, 1971-2041.
[5] RYZHOVA E V, VOS R M, ALBRIGHT A V, et al. Annexin 2:A novel human immunodeficiency virus type 1Gag binding protein involved in replication in monocytederived macrophages[J]. J Virol, 2006, 80(6):2694-2704.
[6] LI J, GUO D, HUANG L, et al.The interaction between host Annexin A2 and viral Nsp9 is beneficial for replication of porcine reproductive and respiratory syndrome virus[J].Virus Res, 2014, 189:106-113.
[7] MEI M, YE J, QIN A, et al. Identification of novel viral receptors with cell line expressing viral receptor-binding protein[J]. Sci Rep, 2015, 5:7935.
[8] MA Y, SUN J, GU L, et al. Annexin A2(ANXA2)interacts with nonstructural protein 1 and promotes the replication of highly pathogenic H5N1 avian influenza virus[J].BMC Microbiol, 2017, 17(1):191.
[9] WU Y, PENG C, XU L, et al. Proteome dynamics in primary target organ of infectious bursal disease virus[J].Proteomics, 2012, 12(11):1844-1859.
[10] REN X, XUE C, KONG Q, et al. Proteomic analysis of purified Newcastle disease virus particles[J]. Proteome Sci, 2012, 10(1):32.
[11] LEBOUDER F, MORELLO E, RIMMELZWAAN G F, et al. AnnexinⅡincorporated into influenza virus particles supports virus replication by converting plasminogen into plasmin[J]. J Virol, 2008, 82(14):6820-6828.
[12] SHENG C, LIU X, JIANG Q, et al. Annexin A2 is involved in the production of classical swine fever virus infectious particles[J]. J Gen Virol, 2015, 96(5):1027-1032.
[13] ZHANG C, XUE C, LI Y, et al. Profiling of cellular proteins in porcine reproductive and respiratory syndrome virus virions by proteomics analysis[J]. Virol J, 2010, 7:242.
[14] RESCHER U, GERKE V. Annexins-unique membrane binding proteins with diverse functions[J]. J Cell Sci,2004, 117(13):2631-2639.
[15] BABIYCHUK E B, DRAEGER A. Regulation of ecto-5'-nucleotidase activity via Ca2+-dependent, annexin 2-mediated membrane rearrangement?[J]. Biochem Soc Trans,2006, 34(3):374-376.
[16] JOST M, GERKE V. Mapping of a regulatory important site for protein kinase C phosphorylation in the N-terminal domain of annexinⅡ[J]. Biochim Biophys Acta,1996, 1313(3):283-289.
[17] SHAO C, ZHANG F, KEMP M M, et al. Crystallographic analysis of calcium-dependent heparin binding to annexin A2[J]. J Biol Chem, 2006, 281(42):31689-31695.
[18] MA G, GREENWELL-WILD T, LEI K, et al. Secretory leukocyte protease inhibitor binds to annexinⅡ, a cofactor for macrophage HIV-1 infection[J]. J Exp Med,2004, 200(10):1337-1346.
[19] PELCHEN-MATTHEWS A, KRAMER B, MARSH M. Infectious HIV-1 assembles in late endosomes in primary macrophages[J]. J Cell Biol, 2003, 162(3):443-455.
[20] BEATON A R, RODRIGUEZ J, REDDY Y K, et al. The membrane trafficking protein calpactin forms a complex with bluetongue virus protein NS3 and mediates virus release[J]. Proc Natl Acad Sci, 2002, 99(20):13154-13159.
[21] YANG Z, SHI Z, GUO H, et al. Annexin 2 is a host protein binding to classical swine fever virus E2 glycoprotein and promoting viral growth in PK-15 cells[J]. Virus Res, 2015, 201:16-23.
[22] YANG S L, CHOU Y T, WU C N, et al. AnnexinⅡbinds to capsid protein VP1 of enterovirus 71 and enhances viral infectivity[J]. J Virol, 2011, 85(22):11809-11820.
[23] SU H, YANG X, WANG S, et al. Effect of annexinⅡ-mediated conversion of plasmin from plasminogen on airborne transmission of H9N2 avian influenza virus[J]. Vet Microbiol, 2018, 223:100-106.