采用杆状病毒/昆虫表达系统纯化蛋白制备抗hUTP14a多克隆抗体
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摘要
为制备兔抗人hUTP14a多克隆抗体并鉴定其抗体的特异性,本研究通过杆状病毒/昆虫表达系统制备并纯化Flag-hUTP14a-his蛋白,免疫新西兰家兔。ELISA方法检测免疫兔的抗血清滴度达到1∶10~5(体积比)时取血清,并通过Protein A免疫亲和层析柱纯化抗体。在人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)中过表达Flag-hUTP14a,或用小干扰RNA(small interference RNA, siRNA)沉默内源的hUTP14a蛋白表达后,提取细胞总蛋白质。经Western印迹分析制备的多克隆抗体的特异性;通过细胞免疫化学染色、细胞免疫荧光、免疫组织化学染色和免疫沉淀(immunoprecipitation, IP)实验对hUTP14a抗体的特异性进行鉴定。研究证实,制备的抗hUTP14a多克隆抗体能够特异性识别内源及外源表达的hUTP14a蛋白。该抗体可以用于细胞免疫荧光、细胞免疫化学染色、免疫组织化学染色、Western印迹及免疫沉淀等技术,为进一步研究hUTP14a的生物学功能提供了特异性抗体。
To prepare specific antibody against hUTP14 a, Flag-hUTP14 a-his protein was expressed and purified from the baculovirus/insect expression system. The purified Flag-hUTP14 a-his protein was used to immunize New Zealand rabbits. The sera were collected from the immunized rabbits when the titer of the anti-sera reached 1∶10~5(volume ratio) measured by ELISA. IgG from the rabbit anti-sera were purified by protein A immuno-affinity chromatography. Western blot was performed with the purified IgG from the anti-sera on the cell lysates of human umbilical vein endothelial cells(HUVEC) transfected with either Flag-hUTP14 a or hUTP14 a-specific siRNA. The purified IgG specifically recognizes endogenous hUTP14 a and ectopic Flag-hUTP14 a, demonstrating that polyclonal anti-hUTP14 a antibody were obtained. It was confirmed that the anti-hUTP14 a antibody could be used in the immunocytochemistry, immunofluorescence(IF), immunohistochemistry staining(IHC) and immunoprecipitation(IP). We have thus obtained a specific rabbit anti-human hUTP14 a polyclonal antibody that can be used in the further study of the biological functions of hUTP14 a.
To prepare specific antibody against hUTP14 a, Flag-hUTP14 a-his protein was expressed and purified from the baculovirus/insect expression system. The purified Flag-hUTP14 a-his protein was used to immunize New Zealand rabbits. The sera were collected from the immunized rabbits when the titer of the anti-sera reached 1∶10~5(volume ratio) measured by ELISA. IgG from the rabbit anti-sera were purified by protein A immuno-affinity chromatography. Western blot was performed with the purified IgG from the anti-sera on the cell lysates of human umbilical vein endothelial cells(HUVEC) transfected with either Flag-hUTP14 a or hUTP14 a-specific siRNA. The purified IgG specifically recognizes endogenous hUTP14 a and ectopic Flag-hUTP14 a, demonstrating that polyclonal anti-hUTP14 a antibody were obtained. It was confirmed that the anti-hUTP14 a antibody could be used in the immunocytochemistry, immunofluorescence(IF), immunohistochemistry staining(IHC) and immunoprecipitation(IP). We have thus obtained a specific rabbit anti-human hUTP14 a polyclonal antibody that can be used in the further study of the biological functions of hUTP14 a.
引文
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[13] Uchida C, Miwa S, Kitagawa K, et al. Enhanced Mdm2 activity inhibits pRB function via ubiquitin- dependent degradation [J]. EMBO J, 2005, 24 (1): 160-169
[14] Nuc P, Nuc K. Recombinant protein production in Escherichia coli [J]. Postepy Biochem, 2006, 52(4): 448-456
[15] 张鹤铭, 焦雪苗, 甘龙站, 等. 重组人白血病抑制因子的表达、包涵体复性、纯化及其活性[J]. 应用与环境生物学报 (Zhang HM, Jiao XM, Gan LZ, et al. Expression, inclusion body renaturation, purification, and activity of recombinant human leukemia inhibitory factor[J]. Chin J Appl Environ Biol 2018, 24(1): 0001-0006
[16] 刘慧,张守涛,郭亚楠. 人P53的原核表达及包涵体复性研究 [J]. 河南师范大学学报(自然科学版)(Liu H, Zhang ST, Guo YN. Expression, purification and renaturation of human P53 [J]. J Henan Normal Univ (Nat Sci Ed), 2016, 44(4): 112-117
[17] Murphy CI, McIntire JR, Davis DR, et al. Enhanced expression, secretion, and large-scale purification of recombinant HIV-1 gp120 in insect cell using the baculovirus egt and p67 signal peptides [J]. Protein Expr Purif, 1993, 4(5): 349-57
[18] Ailor E, Betenbaugh MJ. Modifying secretion and post-translational processing in insect cells [J]. Curr Opin Biotechnol, 1999, 10(2):142-145
[2] Liu X, Cai S, Zhang C, et al. Deacetylation of NAT10 by Sirt1 promotes the transition from rRNA biogenesis to autophagy upon energy stress [J]. Nucleic Acids Res, 2018, 46(18): 9601-9616
[3] Hu L, Wang J, Liu Y, et al. A small ribosomal subunit (SSU) processome component, the human U3 protein 14A (hUTP14A) binds p53 and promotes p53 degradation [J]. J Biol Chem, 2011, 286(4): 3119-3128
[4] Ma T, Lu C, Guo Y, et al. Human U3 protein 14a plays an anti-apoptotic role in cancer cells [J]. Biol Chem, 2017, 398(11): 1247-1257
[5] Liu H, Wang J, Liu Y, et al. Human U3 protein14a is a novel type ubiquitin ligase that binds RB and promotes RB degradation depending on a leucine-rich region [J]. Biochim Biophys Acta Mol Cell Res, 2018, 1865(11 Pt A): 1611-1620
[6] Zhang J, Ren P, Xu D, et al. Human UTP14a promotes colorectal cancer progression by forming a positive regulation loop with c-Myc [J]. Cancer Lett, 2019, 440-441: 106-115
[7] 郑宗方, 韩巍, 何其华, 等. KIAA0649多克隆抗体的制备及其细胞内定位[J]. 北京大学学报(医学版) (Zheng ZF, Han W, He QH, et al. Preparation of the polyclonal antibody against KIAA0649 and its cellular localization [J]. J Peking Univ Health Sci), 2009, 41(6): 613-619
[8] 杨蓉, 谢忠平, 龙润乡, 等. Protein A/G亲和层析在HCV抗体纯化中的应用效果[J]. 医学研究杂志 (Yang R, Xie ZP, Long RX, et al. Application effect of proteinA/G affinity chromatograph in Heapatitis C virus antibody purification [J]. J Med Res), 2010, 39(1): 24-26
[9] Sdek P, Ying H, Chang DL, et al. MDM2 promotes proteasome-dependent ubiquitin-independent degradation of retinoblastoma protein [J]. Mol Cell, 2005, 20(5): 699-708
[10] Scheffner M, Huibregtse JM, Vierstra RD, et al. The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53 [J]. Cell, 1993, 75 (3): 495-505
[11] Burkhart DL, Sage J. Cellular mechanisms of tumour suppression by the retinoblastoma gene [J]. Nat Rev Cancer, 2008, 8(9): 671-682
[12] Ji P, Jiang H, Rekhtman K, et al. An Rb-Skp2-p27 pathway mediates acute cell cycle inhibition by Rb and is retained in a partial-penetrance Rb mutant [J]. Mol Cell, 2004, 16(1): 47-58
[13] Uchida C, Miwa S, Kitagawa K, et al. Enhanced Mdm2 activity inhibits pRB function via ubiquitin- dependent degradation [J]. EMBO J, 2005, 24 (1): 160-169
[14] Nuc P, Nuc K. Recombinant protein production in Escherichia coli [J]. Postepy Biochem, 2006, 52(4): 448-456
[15] 张鹤铭, 焦雪苗, 甘龙站, 等. 重组人白血病抑制因子的表达、包涵体复性、纯化及其活性[J]. 应用与环境生物学报 (Zhang HM, Jiao XM, Gan LZ, et al. Expression, inclusion body renaturation, purification, and activity of recombinant human leukemia inhibitory factor[J]. Chin J Appl Environ Biol 2018, 24(1): 0001-0006
[16] 刘慧,张守涛,郭亚楠. 人P53的原核表达及包涵体复性研究 [J]. 河南师范大学学报(自然科学版)(Liu H, Zhang ST, Guo YN. Expression, purification and renaturation of human P53 [J]. J Henan Normal Univ (Nat Sci Ed), 2016, 44(4): 112-117
[17] Murphy CI, McIntire JR, Davis DR, et al. Enhanced expression, secretion, and large-scale purification of recombinant HIV-1 gp120 in insect cell using the baculovirus egt and p67 signal peptides [J]. Protein Expr Purif, 1993, 4(5): 349-57
[18] Ailor E, Betenbaugh MJ. Modifying secretion and post-translational processing in insect cells [J]. Curr Opin Biotechnol, 1999, 10(2):142-145