刘兰军,何太平,杨春,雍雪飞,何敏,葛永红
LIU Lan-jun,HE Tai-ping,YANG Chun,et al (Chengdu Institute of Biological Products,Chengdu 610023,China)

• 1:成都蓉生药业有限责任公司抗体开发研究室
• 2:成都蓉生药业有限责任公司抗体开发研究室
• 3:成都蓉生药业有限责任公司抗体开发研究室
• 4:成都蓉生药业有限责任公司抗体开发研究室

摘要(Abstract)：

目的克隆人巨细胞病毒包膜糖蛋白gB/AD-1片段并构建其原核表达系统。方法用PCR法从人巨细胞病毒AD169株基因组DNA中扩增gB/AD-1片段并克隆至pGEM-T载体,酶切后,亚克隆至表达载体pET-15b,构建重组表达质粒pET-15b/gB/AD-1,并转化大肠杆菌,IPTG诱导表达。表达产物经金属螯合层析一步纯化,并用Westernblot鉴定。结果gB/AD-1蛋白表达量达到35%。表达产物经纯化后,纯度大于95%。经Westernblot检测,表达产物与CMV阳性人血清发生特异性反应。结论已成功构建重组表达载体pET-15b/gB/AD-1,并在大肠杆菌中高水平表达gB/AD-1。
Objective To clone the gene fragment gB/AD-1 encoding the glycoprotein of human cytomegalovirus(HCMV) and construct its prokaryotic expression system.Methods Amplify gB/AD-1 gene fragment from the genomic DNA of HCMV AD169 strain and clone into vector pGEM-T,then,after identification by sequencing,subclone to expression vector pET-15b. Transform the constructed recombinant plasmid pET-15b/gB/AD-1 to E.coli for expression of gB/AD-1 under induction of IPTG.Purify the expressed product by one-step immobilized metal ion affinity chromatography(IMAC) and identify by Western blot.Results The expressed gB/AD-1 contained 35% of total somatic protein.The purity of expressed product after purification was more than 95%.Western blot showed specific reaction of the expressed product with CMV antibody-positive human serum.Conclusion The recombinant plasmid pET-15b/gB/AD-1 was successfully constructed,and gB/AD-1 was highly expressed in E.coli.

关键词(KeyWords)： 人巨细胞病毒;包膜糖蛋白;抗原决定簇
Human cytomegalovirus;Glycoprotein;Antigenic domain

Abstract:

Keywords:

基金项目(Foundation):

作者(Author): 刘兰军,何太平,杨春,雍雪飞,何敏,葛永红
LIU Lan-jun,HE Tai-ping,YANG Chun,et al (Chengdu Institute of Biological Products,Chengdu 610023,China)

DOI: 10.13200/j.cjb.2006.06.24.liulj.006

参考文献(References)：

• [1]Weill D,Lock BJ,Wewers DL,et al.Combination prophylaxis with ganciclovir and cytomegalovirus(CMV)immune globulin after lung transplantation:effective CMV prevention following daclizumab in-duction.Am J Transplant,2003,3(4):492-496.
• [2]Marshall GS,Rabalais GP,Britt WJ,et al.Antibodies to recombi-nant-derived glycoprotein B after natural human cytomegalovirus in-fection correlate with neutralizing activity.J Infect Dis,1992,165(2):381-384.
• [3]Kropff B,Landini M,Mach M,et al.An ELISA using recombinant proteins for the detection of neutralizing antibodies against human cytomegalovirus.J Med Virol,1993,39:187-195.
• [4]Laemmli UK.Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature,1970,227:680-685.
• [5]Chou S,Marousek G,Parenti DM,et al.Mutation in regionⅢof the DNApolymerase gene conferring foscarnet resistance in cytomeg-alovirus isolates from3subjects receiving prolonged antiviral thera-py.J Infect Dis,1998,178(2):526-530.
• [6]Marshall GS,Rabalais GP,Stout GG,et al.Antibodies to recombi-nant-derived glycoprotein B after natural human cytomegalovirus in-fection correlate with neutralizing activity.J Infect Dis,1992,165(2):381-384.
• [7]Britt WJ,Vugler L,Butfiloski EJ.Cell surface expression of human cytomegalovirus(HCMV)gp55-116(gB):use of HCMV-recombi-nant vaccinia virus-infected cells in analysis of the human neutrali-zing antibody response.J Virol,1990,64(3):1079-1085.
• [8]Britt WJ,Mach M.Human cytomegalovirus glycoproteins.Intervir-ology,1996,39:401-412.
• [9]Britt WJ,Jarvis MA,Drummond DD,et al.Antigenic domain1is required for oligomerization of human cytomegalovirus glycoprotein B.J Virol,2005,79(7):4066-4079.