马立克氏病毒被膜蛋白VP16(UL48)的表达及多克隆抗体的制备
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摘要
UL48蛋白是MDV血清Ⅰ型(MDV-Ⅰ)复制所必需,其与MDV编码的另外一种具有去泛素化酶活性的被膜蛋白UL36以及其他被膜蛋白相互作用,为探究MDV编码的UL48与UL36互作在马立克氏病(MD)肿瘤发生机制中作用,本试验制备了致病型强毒MDV-Ⅰ编码的UL48的抗体。从MDV-Ⅰ基因组中克隆了UL48基因,并将测序正确的UL48基因分别亚克隆到pTYB1和pGEX-4T3原核表达载体,构建成pTYB1-UL48和pGEX4T3-UL48重组质粒。将两重组质粒分别转化到BL21(DE3)E.coli中,分别进行IPTG诱导表达,其中pTYB1-UL48表达的融合蛋白Intein-UL48应用Chitin-Sepharose进行亲和层析纯化,将纯化后的融合蛋白Intein-UL48作为免疫大白兔制备多克隆抗体,其中Intein标签有利于提高UL48蛋白的可溶性并提高抗体效价,pGEX4T3-UL48表达纯化的融合蛋白GST-UL48应用凝血酶进行切割得到UL48蛋白,以此为包被抗原,用于间接ELISA和Western blot抗体效价检测和特异性鉴定。结果显示该抗体效价为1:512 000,成功制备特异性的UL48抗体。
UL48plays essential role in replication of MDV genome and interacts with UL36 as well as other MDV tegument proteins.To investigate the interaction between UL48 and UL36during MDV oncogenisis,antibody against UL48 was prepared and characterized in current study.UL48 gene was amplified from MDV-Ⅰ genome and then subcloned into pTYB1 and pGEX-4T3 vectors for UL48 expression with induction of IPTG in BL21(DE3)E.coli cells.Chitin-sepharose and Glutathion-sepharose were,respectively,used to purify fusion protein intein-UL48 and GST-UL48.Four subcutaneous injections of intein-UL48 fusion protein were done on the lower back and the thigh of rabbit and then other three injections with an interval 10 days.The titer of antibody was measured by the sandwich ELISA with UL48 protein isolated from GST-UL48 after cleavage of thrombin.Western blot was carried out for specificity analysis of antibody against UL48 protein.The results suggested that UL48 antibody was succesfully prepared,and its titer was 1∶512 000.
UL48plays essential role in replication of MDV genome and interacts with UL36 as well as other MDV tegument proteins.To investigate the interaction between UL48 and UL36during MDV oncogenisis,antibody against UL48 was prepared and characterized in current study.UL48 gene was amplified from MDV-Ⅰ genome and then subcloned into pTYB1 and pGEX-4T3 vectors for UL48 expression with induction of IPTG in BL21(DE3)E.coli cells.Chitin-sepharose and Glutathion-sepharose were,respectively,used to purify fusion protein intein-UL48 and GST-UL48.Four subcutaneous injections of intein-UL48 fusion protein were done on the lower back and the thigh of rabbit and then other three injections with an interval 10 days.The titer of antibody was measured by the sandwich ELISA with UL48 protein isolated from GST-UL48 after cleavage of thrombin.Western blot was carried out for specificity analysis of antibody against UL48 protein.The results suggested that UL48 antibody was succesfully prepared,and its titer was 1∶512 000.
引文
[1]邱亚峰,葛菲菲,冯秀丽,等.马立克氏病病毒(MDV)衣壳蛋白VP16的融合表达、纯化及抗体的制备[J].农业生物技术学报,2006,14(4):530-532.
[2]牛成明,韩凌霞.鸡马立克氏病毒抗性相关基因研究进展[J].中国比较医学杂志,2009,19(4):67-70.
[3]罗满林,蔡宝祥,陈溥言.马立克氏病病毒的分子生物学研究进展[J].中国病毒学,1994(2):87-94.
[4]DORANGE F,TISCHER B K,VAUTHEROT J F,et al.Characterization of Marek's disease virus serotype 1(MDV-1)deletion mutants that lack UL46to UL49genes:MDV-1 UL49,encoding VP22,is indispensable for virus growth[J].J Virol,2002,76(4):1959-1970.
[5]DORANGE F,EL MEHDAOUI S,PICHON C,et al.Marek's disease virus(MDV)homologues of herpes simplex virus type 1 UL49(VP22)and UL48(VP16)genes:high-level expression and characterization of MDV-1VP22and VP16[J].J Gen Virol,2000,81(9):2219-2230.
[6]FUCHS W,GRANZOW H,KLUPP B G,et al.The UL48tegument protein ofpseudorabies virus is critical for intracytoplasmic assembly of infectious virions[J].J Virol,2002,76(13):6729-6742.
[7]方六荣,牛传双,肖少波,等.含伪狂犬病毒完整UL49基因及部分UL48基因片段的克隆与序列分析[J].华中农业大学学报,2002,21(4):306-310.
[8]赖晓芳,蔡发国,王炜军,等.萝卜块根中两个具溶菌酶活性的几丁质结合蛋白的纯化及其特性[J].植物生理与分子生物学学报,2006,32(4):445-450.
[9]李妍,姜茵,奚月,等.幽门螺杆菌Catalas/GST融合蛋白的可溶性表达及凝血酶柱上切割GST标签[J].现代预防医学,2012,39(21):110-112.
[10]郑海南,邓晨,王梦云,等.马立克病毒UL36~(USP)蛋白的表达及其抗体制备[J].中国兽医学报,2014,34(5):712-716.
[11]张苗苗,陶章生,陈婷婷,等.水稻纤维素合酶多克隆抗体的制备和鉴定[J].华中农业大学学报,2011,30(4):393-397.
[12]邓晨,于佩峰,张鑫,等.应用Intein融合的鸡IL-2蛋白制备多克隆抗体[J].吉林农业大学学报,2015,37(3):368-374.
[13]HELFERICH D,VEITS J,METTENLEITER T C,et al.Identification of transcripts and protein products of the UL31,UL37,UL46,UL47,UL48,UL49and US4gene homologues of avian infectious laryngotracheitis virus[J].J Gen Virol,2007,88(3):719-731.
[14]JAROSINSKI K W,VAUTHEROT J F.Differential expression of Marek's disease virus(MDV)late proteins during in vitro and in situ replication:Role for pUL47in regulation of the MDV UL46#UL49gene locus[J].Virology,2015,484:213-226.
[15]毕智丽.pTYB11载体的应用及其重组子破菌条件的研究[J].科技信息,2012(5):34.
[2]牛成明,韩凌霞.鸡马立克氏病毒抗性相关基因研究进展[J].中国比较医学杂志,2009,19(4):67-70.
[3]罗满林,蔡宝祥,陈溥言.马立克氏病病毒的分子生物学研究进展[J].中国病毒学,1994(2):87-94.
[4]DORANGE F,TISCHER B K,VAUTHEROT J F,et al.Characterization of Marek's disease virus serotype 1(MDV-1)deletion mutants that lack UL46to UL49genes:MDV-1 UL49,encoding VP22,is indispensable for virus growth[J].J Virol,2002,76(4):1959-1970.
[5]DORANGE F,EL MEHDAOUI S,PICHON C,et al.Marek's disease virus(MDV)homologues of herpes simplex virus type 1 UL49(VP22)and UL48(VP16)genes:high-level expression and characterization of MDV-1VP22and VP16[J].J Gen Virol,2000,81(9):2219-2230.
[6]FUCHS W,GRANZOW H,KLUPP B G,et al.The UL48tegument protein ofpseudorabies virus is critical for intracytoplasmic assembly of infectious virions[J].J Virol,2002,76(13):6729-6742.
[7]方六荣,牛传双,肖少波,等.含伪狂犬病毒完整UL49基因及部分UL48基因片段的克隆与序列分析[J].华中农业大学学报,2002,21(4):306-310.
[8]赖晓芳,蔡发国,王炜军,等.萝卜块根中两个具溶菌酶活性的几丁质结合蛋白的纯化及其特性[J].植物生理与分子生物学学报,2006,32(4):445-450.
[9]李妍,姜茵,奚月,等.幽门螺杆菌Catalas/GST融合蛋白的可溶性表达及凝血酶柱上切割GST标签[J].现代预防医学,2012,39(21):110-112.
[10]郑海南,邓晨,王梦云,等.马立克病毒UL36~(USP)蛋白的表达及其抗体制备[J].中国兽医学报,2014,34(5):712-716.
[11]张苗苗,陶章生,陈婷婷,等.水稻纤维素合酶多克隆抗体的制备和鉴定[J].华中农业大学学报,2011,30(4):393-397.
[12]邓晨,于佩峰,张鑫,等.应用Intein融合的鸡IL-2蛋白制备多克隆抗体[J].吉林农业大学学报,2015,37(3):368-374.
[13]HELFERICH D,VEITS J,METTENLEITER T C,et al.Identification of transcripts and protein products of the UL31,UL37,UL46,UL47,UL48,UL49and US4gene homologues of avian infectious laryngotracheitis virus[J].J Gen Virol,2007,88(3):719-731.
[14]JAROSINSKI K W,VAUTHEROT J F.Differential expression of Marek's disease virus(MDV)late proteins during in vitro and in situ replication:Role for pUL47in regulation of the MDV UL46#UL49gene locus[J].Virology,2015,484:213-226.
[15]毕智丽.pTYB11载体的应用及其重组子破菌条件的研究[J].科技信息,2012(5):34.