风疹病毒E1重组蛋白及其抗原肽在大肠杆菌中的表达
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摘要
目的
利用重叠PCR合成风疹病毒E1全长基因,同时扩增E1主要抗原表位的基因序列,分别构建它们的原核表达载体并表达蛋白,为研发高灵敏度风疹ELISA诊断试剂奠定基础。
方法
对风疹E1基因进行生物信息学分析,根据大肠杆菌密码子偏爱性对其密码子进行优化；设计多对寡核苷酸引物,以重叠PCR法分别合成该基因的3个片段,再用酶切连接法将各段拼接成全长为1 443 bp的RV E1,将E1基因的全长序列克隆导入原核表达载体pET32a获得pET32-RV E1克隆并测序；选取E1全长基因中195 aa-305 aa抗原肽基因序列进行扩增,再以酶切连接的方法将合成的抗原肽序列克隆导入原核表达载体pET32a,获得重组质粒载体pET32-E1epitope测序鉴定。将重组原核表达载体pET32-RV El和pET32-E1epitope在大肠杆菌BL21(DE3)表达株中利用IPTG诱导蛋白表达并优化蛋白表达量, SDS-PAGE和Western blot分析表达产物。
结果
1.分别进行了8轮、5轮和6轮的重叠PCR扩增,合成风疹E1全长基因3个片段；以酶切连接法将3个片段拼接成全长E1基因并克隆入pET32a,构建载体pET32-RV E1,PCR、酶切和测序鉴定结果表明,合成的E1基因大小、序列与预期相符；构建获得重组质粒pET32-RV E1。在37℃下用终浓度为1 mmol/L的IPTG诱导蛋白表达,Western blot检测到预期的蛋白条带；
2.以PCR扩增成功获得风疹E1抗原肽基因并构建重组质粒pET32-El epitope,测序结果正确；在37℃下以终浓度为1 mmol/L IPTG诱导抗原肽表达,SDS-PAGE和Western blot检测到预期的蛋白条带；且以IPTG终浓度为0.1 mmol/L诱导6小时后表达量达到最高。
结论
成功合成了密码子优化的风疹病毒E1基因并构建其重组质粒pET32-RV E1,表达完整E1蛋白；同时成功扩增风疹病毒E1抗原肽段基因,构建其原核表达载体pET32-E1epitope并表达该抗原肽。
Objectives
To research efficient recombinant antigens for serological assays for rubella virus infection, full-length rubella virus E1 gene was synthesized with overlapping PCR-Restriction enzyme ligation method and major E1 epitope gene was amplified by PCR; Full-length E1 gene and E1 epitope gene were constructed into prokaryotic vectors for expression of recombinant antigen, respectively.
Methods
Bioinformatic analysis of E1 gene was done and the E1 sequence containing Escherichia coli preferred codons was designed. Two restriction enzymes were used to cut the full-length sequence of E1 to generate three fragments. Which were then amplified, respectively. With overlapping PCR using multiple pairs of oligonucleotide primers, a full-length of E1 sequence was assembled via restriction enzyme ligation. The full-length of E1 sequence was cloned into expression vector pET32a. The recombinant plasmid pET32-RV E1 was confirmed by PCR, restriction enzyme digestion and DNA sequencing. The gene of epitope,195-305 amino acid residues of the rubella virus E1 glycoprotein, was amplified from new synthesized E1 sequence, and cloned into expression vector pET32a to construct recombinant plasmid pET32-E1epitope-The expression of the two recombinant proteins was induced by IPTG, and analyzed by SDS-PAGE and Western blotting.
Results
1. The three fragments were amplified, and verified by DNA sequencing. The full-length sequence was inserted into plasmid pET32a which was confirmed as the designed one. The recombinant protein was expressed with IPTG inducement and identified by western blotting.
2. The desired epitope gene fragment was amplified with PCR and confirmed with DNA sequencing. The sequence was cloned into plasmid pET32a, which was confirmed as the desired with PCR, restriction enzyme digestion and DNA sequencing. Western blotting showed the expression of recombinant protein with IPTG inducement. The recombinant protein of high level was expressed at 6 hours after induced with 0.1 mmol/L IPTG.
Conclusions
The rubella virus E1 gene sequence containing Escherichia coli preferred codons was synthesized with overlapping PCR-Restriction enzyme ligation method, and the plasmid pET32-RV E1 was constructed. The plasmid pET32-E1epitope for E1 epitope gene was constructed too. The recombinant full-length El protein and E1 epitope was expressed.
利用重叠PCR合成风疹病毒E1全长基因,同时扩增E1主要抗原表位的基因序列,分别构建它们的原核表达载体并表达蛋白,为研发高灵敏度风疹ELISA诊断试剂奠定基础。
方法
对风疹E1基因进行生物信息学分析,根据大肠杆菌密码子偏爱性对其密码子进行优化；设计多对寡核苷酸引物,以重叠PCR法分别合成该基因的3个片段,再用酶切连接法将各段拼接成全长为1 443 bp的RV E1,将E1基因的全长序列克隆导入原核表达载体pET32a获得pET32-RV E1克隆并测序；选取E1全长基因中195 aa-305 aa抗原肽基因序列进行扩增,再以酶切连接的方法将合成的抗原肽序列克隆导入原核表达载体pET32a,获得重组质粒载体pET32-E1epitope测序鉴定。将重组原核表达载体pET32-RV El和pET32-E1epitope在大肠杆菌BL21(DE3)表达株中利用IPTG诱导蛋白表达并优化蛋白表达量, SDS-PAGE和Western blot分析表达产物。
结果
1.分别进行了8轮、5轮和6轮的重叠PCR扩增,合成风疹E1全长基因3个片段；以酶切连接法将3个片段拼接成全长E1基因并克隆入pET32a,构建载体pET32-RV E1,PCR、酶切和测序鉴定结果表明,合成的E1基因大小、序列与预期相符；构建获得重组质粒pET32-RV E1。在37℃下用终浓度为1 mmol/L的IPTG诱导蛋白表达,Western blot检测到预期的蛋白条带；
2.以PCR扩增成功获得风疹E1抗原肽基因并构建重组质粒pET32-El epitope,测序结果正确；在37℃下以终浓度为1 mmol/L IPTG诱导抗原肽表达,SDS-PAGE和Western blot检测到预期的蛋白条带；且以IPTG终浓度为0.1 mmol/L诱导6小时后表达量达到最高。
结论
成功合成了密码子优化的风疹病毒E1基因并构建其重组质粒pET32-RV E1,表达完整E1蛋白；同时成功扩增风疹病毒E1抗原肽段基因,构建其原核表达载体pET32-E1epitope并表达该抗原肽。
Objectives
To research efficient recombinant antigens for serological assays for rubella virus infection, full-length rubella virus E1 gene was synthesized with overlapping PCR-Restriction enzyme ligation method and major E1 epitope gene was amplified by PCR; Full-length E1 gene and E1 epitope gene were constructed into prokaryotic vectors for expression of recombinant antigen, respectively.
Methods
Bioinformatic analysis of E1 gene was done and the E1 sequence containing Escherichia coli preferred codons was designed. Two restriction enzymes were used to cut the full-length sequence of E1 to generate three fragments. Which were then amplified, respectively. With overlapping PCR using multiple pairs of oligonucleotide primers, a full-length of E1 sequence was assembled via restriction enzyme ligation. The full-length of E1 sequence was cloned into expression vector pET32a. The recombinant plasmid pET32-RV E1 was confirmed by PCR, restriction enzyme digestion and DNA sequencing. The gene of epitope,195-305 amino acid residues of the rubella virus E1 glycoprotein, was amplified from new synthesized E1 sequence, and cloned into expression vector pET32a to construct recombinant plasmid pET32-E1epitope-The expression of the two recombinant proteins was induced by IPTG, and analyzed by SDS-PAGE and Western blotting.
Results
1. The three fragments were amplified, and verified by DNA sequencing. The full-length sequence was inserted into plasmid pET32a which was confirmed as the designed one. The recombinant protein was expressed with IPTG inducement and identified by western blotting.
2. The desired epitope gene fragment was amplified with PCR and confirmed with DNA sequencing. The sequence was cloned into plasmid pET32a, which was confirmed as the desired with PCR, restriction enzyme digestion and DNA sequencing. Western blotting showed the expression of recombinant protein with IPTG inducement. The recombinant protein of high level was expressed at 6 hours after induced with 0.1 mmol/L IPTG.
Conclusions
The rubella virus E1 gene sequence containing Escherichia coli preferred codons was synthesized with overlapping PCR-Restriction enzyme ligation method, and the plasmid pET32-RV E1 was constructed. The plasmid pET32-E1epitope for E1 epitope gene was constructed too. The recombinant full-length El protein and E1 epitope was expressed.
引文
[1]Ingalls TH, Babbott FL, Hampson KW, et al. Rubella:its epidemiology and teratology[J]. Am J MedSei,1960,239:363-383.
[2]Frey TK. Molecular biology of rubella virus[J]. Adv Virus Res,1994,44:69-160.
[3]Katow S. Rubella virus. Nippon Rinsho,l999,57:313-5.
[4]Domingnez G., Wang C.Y., Frey T.K. Sequence of the genome RNA of rubella virus:evidemce for genetic rearrangement during togavirus evolution[J]. Virology,1990,177(2):225-238.
[5]Edmunds WJ, Eerola M, Gay NJ. Modelling rubella in Europe[J]. Epidemiol Infect,2000,125(3):617-634.
[6]Terada K, Niizuma T, Daimon Y, et al. Comparison of cost and benefits of each model for rubella immunization in Japan[J]. Kansenshogaku Zasshi,2000:74(12):1012-1017.
[7]Oker-Blom C, Ulmanen I, Kaariainen L, et al.Rubella virus 40s genome RNA specifies a 24S subgenomic mRNA that encodes for a precursor to structural proteins[J]. J Virol,1984, 49(2):403-408.
[8]Oker-Blom C.The gene order of rubella virus structural proteins is:NH2-C-E2-E1-COOH[J]. J Virol,1984,51(2):354-358.
[9]Edwin D,Michael FI, Martin DR. Virus-encoded proteinases of the Togaviridae [J]. J Gen Virol,1999,80(8):1879-1888.
[10]Hobman TC, ShukinR, Gillam S. Translocation of rubella virus glycoprotein Elinto the endoplasmic reticulum[J].J Virol,1988:62(11):4259-4264.
[11]Qiu Z, Ou D,Hobman-TC. Expression and charaeterization of virus-like particles containing rubella virus structural proteins[J].J Virol,1994,68(6):4086-4091.
[12]Qiu ZY, Frank T, Shirley G. The infuence of N-linked glycosylation on antifenicity and immunogenicity of rubella virus E1 glycoprotein[J]. Virology,1992,190(2):876-881.
[13]Ramanujam M, Hofmann J, Nakhasi HL, et al. Effect of site-directed asparagines to isoleucine substitutions at the N-linked E1 glycosylation sites on rubella virus viability[J]. Virus Res,2001,81(1-2):151-156.
[14]Umino Y.,Sato T.A., Katow S.,et al.Monoclonal antibodies directed to E1 glycoprotein of rubella virus[J].Areh Virol,1985,83:33-42.
[15]Waxiham M.N.,Wolillsky J.S.Detailed immunologic analysis of the structural polypeptides of rubella virus using monoclonal antibodies [J]. Virol,1987,143:153-156.
[16]Terry G..M., Ho Terry L. M., Londesborough P.,et al. Localization of the rubella El epitopes[J]. Arch Virol,1988,98:189-197.
[17]Bosma TJ, Best JM, Corbett KM, et al. Nucleotide sequence analysis of a major antigenic domain of the E1 glycoprotein of 22 rubella virus isolates[J]. J Gen Virol,1996,77 (Pt 10): 2523-2530.
[18]Mitchell L.A., ZhangT., Ho M.et al. Characterization of Rubella specific antibody responses by using a new synthetic peptide basedenzyme linked immunosorbent assay[J]. J.Clin. Microbiology. 1992,30;1841-1847.
[19]Chaye H., Chong P., Tripet B. et al. Localization of the virus neutralizing and hemagglutinin epitopes of E1 glycoprotein of rubella virus[J]. Virology,1992,189:483-492.
[20]Chen M.J., Icenogle J.P., Molecular virology of rubella virus. Rubella Virus, J.Banatvala andC.Peekham[J].Oxford, UK, Elsevier,2007:1-18.
[21]Frey TK, Abernathy ES, Bosma TJ,et al. Molecular analysis of rubella virus epidemiology across three continents, North America, Europe, and Asia,1961-1977 [J]. J Infect Dis, 1998,178(3):642-650.
[22]Katow S, Minahara H, Fukushima M, et al. Molecular epidemiology of rubella by nucleotide sequences of the rubella virus El gene in three East Asian countries[J]. Infec Dis,1997,176(3):606-616.
[23]Best JM, Thomson A, Reynors J,et al. Rubella virus strains show no major antigenic differences[J]. Intervirology,1992,34(3):164-168.
[24]Waxham M.N, Wolinsky J.S.. A Model of the Structural Organization of Rubella Virions[J]. Clin Infect Dis,1985,7 (Suppl 1):133-139.
[25]Dalgarno L., Rice C.M., Sreauss J.H.. Ross River virus 26 s RNA:complete nucleotide sequence and deduced sequence of the encoded structural proteins. [J]. Virology,1983,129:170-187.
[26]Hobman TC, Woodward L, Farquhar MG. Targeting of a heterodimeric membrane protein complex to the Golgi:rubella virus E2 glycoprotein contains a transmembrane Golgi retention signal[J]. Mol Biol Cell,1995,6(1):7-20.
[27]Law LM, Duncan R, Esmaili A,et al. Rubella virus E2 singal petide is required for perinuclear localization of capsid protein and virus assembly[J]. J Virol,2001,75(4):1978-1983.
[28]Parkman PD. Making vaccination policy:the experience with Rubella[J]. Clin Infect Dis,1999,28(12):140-146.
[29]Qiu Z, Hobman TC, McDonald HL, et al. Role of N-linked oligosaccharides in processing and intracellular transport of E2 glycoprotein of Rubella virus[J]. Virology,1992,66(6):3514-3521.
[30]Terry H L.,Terry G.M., Londesborough P. Diagnosis of focusing and peptide mapping[J]. Arch Virol,1982,72:47-54.
[31]Dorsett PH, Miller DC, Green KY, et al. Structure and function of the rubella virus proteins [J]. Rev Infect Dis.,1985,7 (Suppl 1):150-156.
[32]Cusi MG, Metelli R, Valensin PE. Immune responses to wild and vaccine rubella viruses after rubella vaccination[J]. Arch Virol,1989,106(1-2):63-72.
[33]Green KY, Dorsett PH. Rubella virus antigens:localization of epitopes involved in hemagglutination and neutralization by using monoclonal antibodies[J]. J Virol,1986, 57(3):893-898.
[34]王志玉.风疹病毒包膜糖蛋白研究进展[J].国外医学·微生物学分册,1995,18(4)：12-13.
[35]Marr LD, Sanchez A, Frey TK. Efficient in vitro translation and proccssing of rubella virus structural proteins in the presence of microsomes[J]. Virology,1991,180(1):400-405.
[36]Bowden DS, Westaway EG. Rubella virus:structrual and non-structural proteins[J]. J Gen Virol,1984,65(5):933-943.
[37]Liu Z, Yang D, QiuZ, et al. Idnetification of domains in rubella viurs genomic RNA and cpasid protein necessary for specific interaction[J]. J Viorl,1996,70(4):2184-2190.
[38]Law LM, Everitt JC, Beatch MD, etal. Phosphorylation of rubella virus capsid regulates its RNA bingding activity and virus replication[J]. J Virol,2003,77(3):1764-1771.
[39]Tzeng WP, Frey TK.ComPlementation of a deletion in the rubella virus p150 nonstructural protein by theviral capsid protein[J]. J Virol,2003,77(17):9502-9510.
[40]Duncan R, Esmaili A, Law LM, et al. Rubella virus capsid protein induces apoptosis in transfected RK13 cells[J]. Virology,2000,275(1):20-29.
[41]Cusi M.G., Rossolini G.M., Cellesi C.,et al. Antibody response to wild rubella virus structural proteins following immunization witg RA27/3 live attenuated vaccine[J].Arch Virol,1988,101:25-33.
[42]彭文伟主编.现代感染性疾病与传染病学[M].第一版.北京：科学出版社.2000：642-653.
[43]Smith CA, Petty RE, Tingle AJ. Rubella virus and arthlitis[J]. Rheum Dis Clin North Am,1987,13(2):265-274.
[44]Portielje J.E.A., Westendorp R.G.J., Kluin-Nelemans H.C., et al. Morbidity and mortality in adults with idiopathic thrombocytopenic purpura[J]. Blood,2001,97 (9):2549-2554.
[45]Baron M.D., Ebel T., Suomalainen M.Inltracellular transport of rubella virus structural proteins expressed from cloned cDNA[J].JGenVirol,1992,73(5):1073-1086
[46]Ruokonen PC, Metzner S, Ucer A, et al. Intraocular antibody synthesis against rubella virus and other microorganisms in Fuchs' heterochromic cyclitis[J]. Graefes Arch Clin Exp Ophthalmol,2010,248:565-571
[47]Bossy-Wetztel E, Green DR. Apoptosis:check point at the mitochondrial frontier[J].Mutat Res,1999,434(3):243-251.
[48]Robertson S.E., Featherstone D.A., Gacic-Dodo M.,et al. Rubella and congeni-tal rubella syndrome:global update[J]. Rev Panam Salud Publica,2003,14(5):306-315.
[49]Cutts, F.T., Vynnyck E..Modelling the incidence of congenital rubella sydrome in developing countries[J]. Int J Epidemiol,1999,28(6):1176-1184.
[50]Lee J., Bowden D.S..Rubella virus replication and links to teratogenicity[J].Clin Microbiol Rev, 2000,13(4):571-587.
[51]Reece EA, Hobbin JC. Medicine of the fetus&mother,2nded[M]. Philadephia:Lippincott-Raven New York,1999:347-365.
[52]李沪,胡家瑜,陶黎纳,等.先天性风疹综合征流行病学特征与免疫预防策略[J].上海预防医学杂志,2005,17(2)：72-74.
[53]KurodaY, Matsui M. Progressive rubella panencephalitis[J]. Nippon Rinsho,1997, 55(4):922-925.
[54]Webster WS. Teratogen update:congenital rubella[J]. Teratology,1998,58(1):13-23.
[55]Miller E, Cradoek-Wstson JE, Pollock TM. Consequences of confirmed maternal rubella at successive stages of pregnancy [J]. Lancet,1982,302(82):781-784.
[56]Megyeri K, Berenesi K, Halazonetis TD, et al.Involvement of a P53-dependent pathway in rubella virus-induced apoptosis[J]. Viorlogy,1999,259(1):74-84.
[57]Hofmann J, Pletz MW, Liebert UG. Rubella virus-induced cytopathic effect in vitro is caused by apoptosis[J]. J Gen Virol,1999,80(7):1657-1664.
[58]Novitski E. Human Genetics P34[M]. Macmillam publishing Co Inc.1997.
[59]Kono R, Hirayama M, Sugishita C, et al. Epidemiology of Rubella and Congenital Rubella Infection in Japan[J]. Reviews of Infectious Diseases,1985,7:56-63
[60]天津医学院流行病教研室.人类病毒性传染病·流行病学及防治[M].北京：人民卫生出版社,1984：442.
[61]Reef S.E. Redd SB, Abernathy E, et al. The epidemiological profile of rubella and congenital rubella syndromein the United States,1998-2004:the evidence for absence of endemic transmission[J].Clin Infect Dis,2006,43,Suppl 3:126-132.
[62]Icenogle, J.P., Frey TK, Abernathy E, et al..Genetic analysis of rubella viruses found in the United States between 1966 and 2004:evidence that indigenous rubella viruses have been eliminated[J]. Clin Infect Dis,2006,43Suppl 3:133-140.
[63]CDC, Elimination of rubella and congenital rubella syndrome-United Srates, 1969-2004[J].MMWR Morb Mortal Wkly Rep,2005,54(11):279-282.
[64]CDC, Achievements in public health:elimination of rubella and congenital rubella syndrome-US,1969-2004[J]. Ann Pharmacother,2005,39(6):1151-1152.
[65]WHO. Progress towards eliminating rubella and congenital rubella syndrome in the western hemisphere,2003-2008. Wkly Epidemiol Rec,2008,83:395-400.
[66]Peltola H, Jokinen S, Paunio M, et al. Measles, mumps, and rubella in Finland:25 years of a nationwide elimination programme[J]. Lancet Infect Dis.2008,8(12):796-803.
[67]D'Agaro P, Dal Molin G, Zamparo E, et al. Epidemiological and molecular assessment of a rubella outbreak in North-Eastern Italy[J]. J Med Virol.2010,82(11):1976-1982.
[68]Cutts FT, Robertson SE, Samuel R, et al. Control of rubella and congenital rubella syndrome(CRS)in developing countries, part 1:burden of disease from CRS[J]. Bull World Health Organ,1997,75(1):55-68.
[69]樊春祥,梁晓峰,周玉清,等.中国2004-2006年风疹流行病学特征初步分析[J].中国计划免疫,2007,13(5)：457-460.
[70]朱贞,郭学斌,崔爱利,等.中国2007-2008年风疹流行病学和病毒基因特征分析[J].中国疫苗和免疫,2009,15(3)：201-204.
[71]马静,郝利新,罗会明,等.中国2008-2009年风疹流行病学特征分析[J].中国疫苗和免疫,2010,16(4)：322-324.
[72]周先军.1082例妊娠妇女TORCH感染情况调查分析[J].检验医学与临床,2010,7(8)：680-681.
[73]陈世勇,赵真真,阮林松,等.早孕妇女1379例TORCH感染情况分析[J].实用医学杂志,2010,26(4)：708-709.
[74]王丽娟,王志玉.风疹病毒检测方法研究进展[J].中国公共卫生,2008,24(6)：749-751.
[75]Gurtadauria S, Cordoba P, Cuffini C, et al. Cell-fusion assay for the detection of rubella virus in Vero cells[J]. Clin Diagn Virol,1998,10(1):9-16.
[76]Yan H, Li G, Yan H,et al. Establishment and characterization of a novel genetic hybrid cell line for propagation of four pathogens [J]. J Virol Methods,2006,134 (1-2):86-91.
[77]Christopher Mpale PA, Jones CS. Time-resolved fluoromertic irnrnunoassay for rubella antibody-a useful method for serosurveillance studies[J]. Vaccine,2002,20 (9-10):1378-1382.
[78]朱贞,许文波,李聪勇,等.一种新型诊断方法—比色法免疫学实验在检测风疹病毒中的应用研究[J].中国计划免疫,2006,12(6)：489-493.
[79]Perry KR, Brown DW, Parry JV, et al. Detection of measles, mumps, and rubella antibodies in saliva using antibody capture radioimmunoassay [J]. J Med Virol,1993,40(3):235-240.
[80]Voller A, Bidwell DE. A simple method for detecting antibodies to rubella[J]. Br J Exp Pathol, 1975,56(4):338-339.
[81]Morris M, Cohen B,Andrews N,et al. Stability of total and rubella-specific IgG in oral fluid samples:the effect of time and temperature[J]. J Immunol Methods,2002,266 (1-2):111-116.
[82]Akingbade D, Cohen BJ, Brown DW. Detection of low-avidity immunoglobulin G in oral fluid samples:new approach for rubella diagnosis and surveillance [J]. Clin Diagn Lab Immunol,2003,10(1):189-190.
[83]Gay NJ, Vyse AJ, Enquselassie F, et al.Implroving sensitivity of oral fiuid testing in IgG pervalence studies:application of mixture models to a rubella antibody survey[J]. Epidemiol Infect,2003,130(2):285-291.
[84]Allmendinger J., Paradies F., Kamprad M., et al. Determination of Rubella Virus-Specific Cell-Mediated Immunity Using IFNy-ELISpot[J]. Journal of Medical Virology,2010,82:335-340.
[85]Ardizzoni A, Capuccini B, Baschieri MC, et al. A protein microarray immunoassay for the serological evaluation of the antibody response in vertically transmitted infections[J]. Eur J Clin Microbiol Infect Dis.2009,28(9):1067-1075
[86]Li D, Yang H, Zhang WH, et al. A simple parallel analytical method of prenatal screening[J]. Gynecol Obstet Invest,2006,62 (4):220-225.
[87]Tanemura M, Suzmuori K, Yagami Y,et al. Diagnosis of fetal rubella infection with reverse transcription and nested polymerase chain reaction:a study of 34 cases diagnosed in fetuses[J]. Am J Obstet Gynecol,1996,174(2):578-582.
[88]Vyse AJ, Jin L.An RT-PCR assay using oral fluid samples to detect rubella viurs genome for epidemiological surveillnace[J].Mol Cell Probes,2002,16(2):93-97.
[89]Shyamala G,Malathi J,Moses YS,et al. Nested reverse transcription polymerase chain reaction for the detection of rubella virus in clinical specimens[J]. Indian J Med Res,2007,125 (1):73-78.
[90]Cooray S,Warrener L,Jin L. Improved RT-PCR for diagnosis and epidemiological surveillance of rubella[J]. J Clin Virol,2006,35 (1):73-80.
[91]Mosuqera MM, Ory FD,Moreno M,et al.Simultaneous detection of measles viurs,rubella viurs, and parvovirus by using multiplex PCR[J].J Clin Micorbiol,2002.40(1):111-116.
[92]Feng Y, Santibanez S, Appleton H, et al. Application of new assays for rapid confirmation and genotyping of isolates of rubella virus[J]. J Med Virol.2011,83(1):170-177.
[93]Mosquera M M,Sanz JC, Echevarria J E,et al. Diagnostic performance of specific IgM detection and genomic amplification in rubella [J]. Enferm Infect Microbiol Clin,2006,24 (4): 251-253.
[94]Liu Q,Bai Y, Ge Q,et al. Microarray-in-a-tube for detection of multiple viruses[J]. Clin Chem,2007,53(2):188-194.
[95]Maeda H, Kokeguchi S, Fujimoto C, et al. Detection of periodontal pathogen Porphyromonas gingivalis by loop-mediated isothermal amplification method [J]. FEMS Immunol Med Microbiol, 2005,43:233-239.
[96]Nagamine K, Hase T,Notomi T. Accelerated reaction by loop-mediated isothermal amplification using loop primers[J]. Molecular and Cellular Probes,2002,16 (3):223-229.
[97]Mori N,Motegi Y, Shimamura Y, et al. Development of a new method for diagnosis of rubella virus infection by reverse transcription-loop-mediated isothermal amplification[J]. J Clin Microbiol,2006,44 (9):3268-3273.
[98]Song T, Toma C, Nakasone N, et al. Sensitive and rapid detection of Shigella and enteroinvasive Escherichia coli by a loop-mediatied isothermal amplification method [J]. FEMS Microbiol Lett,2005,243:259-263.
[99]胡得意,胡永卫,李晓祺,等.学生风疹发病危险因素的病例对照研究[J].浙江预防医学,2010,22(2)：20.
[100]马岚,曹文正,史红,等.兰州市2005-2008年麻疹、风疹实验室检测结果分析[J].中国初级卫生保健,2010,24(2)：76-77.
[101]Prodromou C. Recursive PCR:a novel technique for total gene synthesis [J].Protein Engineering,1992,5(8):827-829.
[102]王志玉,薛永磊,王小凡,等.风疹病毒J R23株E1包膜糖蛋白基因的表达分析[J].病毒学报,2003,19(3)：278-280.
[103]温红玲,王志玉,宋艳艳,等.风疹病毒J R23株糖蛋白E1在毕赤酵母表达系统中的表达和抗原性分析[J].中华微生物学和免疫学杂志,2005,25(3)：190-193.
[104]Hobman TC, Lundstrom ML, Gillam S. Processing and intracellular transport of rubella virus strucural proteins on COS cells. Virology,1990,178:122-133.
[105]Oker-Blom C, Pettersson RF, Summers MD. Baculovirus polyhedron promoterdirected expression of rubella virus envelope glycoproeins E1 and E2, in spodoptera frugiperda cells[J]. Virology,1989,172:82-91.
[106]Oiu ZY, Frank T, Shirley G. The influence of N-linked glycosylation on the antigenicity of rubella virus E1 glycoprotein[J].Virology,1992,190(2):876-881.
[107]Terry HL, Cohen A. The role of glycosylation on haemagglutination and immunological reactivity of rubella virus[J]. Arch Virol,1984,79:139-146.
[108]朱贵明,陈宏,卢建申,等.应用PCR-酶切连接法合成全长sFatl基因[J].遗传,2006,28(9)：1123-8.
[109]Terry GM, Terry HL, Lonesborough P., et al. A bio-engineered rubella E1 antigen [J]. Arch Virol,1989,104 (1-2):63-75.
[110]Starkey WG, Newcombe J, Corbett KM, et al. Use of rubella virus E1 fusion proteins for detection of rubella virus antibodies[J]. J Clin Microbiol,1995,33(2):270-274.
[2]Frey TK. Molecular biology of rubella virus[J]. Adv Virus Res,1994,44:69-160.
[3]Katow S. Rubella virus. Nippon Rinsho,l999,57:313-5.
[4]Domingnez G., Wang C.Y., Frey T.K. Sequence of the genome RNA of rubella virus:evidemce for genetic rearrangement during togavirus evolution[J]. Virology,1990,177(2):225-238.
[5]Edmunds WJ, Eerola M, Gay NJ. Modelling rubella in Europe[J]. Epidemiol Infect,2000,125(3):617-634.
[6]Terada K, Niizuma T, Daimon Y, et al. Comparison of cost and benefits of each model for rubella immunization in Japan[J]. Kansenshogaku Zasshi,2000:74(12):1012-1017.
[7]Oker-Blom C, Ulmanen I, Kaariainen L, et al.Rubella virus 40s genome RNA specifies a 24S subgenomic mRNA that encodes for a precursor to structural proteins[J]. J Virol,1984, 49(2):403-408.
[8]Oker-Blom C.The gene order of rubella virus structural proteins is:NH2-C-E2-E1-COOH[J]. J Virol,1984,51(2):354-358.
[9]Edwin D,Michael FI, Martin DR. Virus-encoded proteinases of the Togaviridae [J]. J Gen Virol,1999,80(8):1879-1888.
[10]Hobman TC, ShukinR, Gillam S. Translocation of rubella virus glycoprotein Elinto the endoplasmic reticulum[J].J Virol,1988:62(11):4259-4264.
[11]Qiu Z, Ou D,Hobman-TC. Expression and charaeterization of virus-like particles containing rubella virus structural proteins[J].J Virol,1994,68(6):4086-4091.
[12]Qiu ZY, Frank T, Shirley G. The infuence of N-linked glycosylation on antifenicity and immunogenicity of rubella virus E1 glycoprotein[J]. Virology,1992,190(2):876-881.
[13]Ramanujam M, Hofmann J, Nakhasi HL, et al. Effect of site-directed asparagines to isoleucine substitutions at the N-linked E1 glycosylation sites on rubella virus viability[J]. Virus Res,2001,81(1-2):151-156.
[14]Umino Y.,Sato T.A., Katow S.,et al.Monoclonal antibodies directed to E1 glycoprotein of rubella virus[J].Areh Virol,1985,83:33-42.
[15]Waxiham M.N.,Wolillsky J.S.Detailed immunologic analysis of the structural polypeptides of rubella virus using monoclonal antibodies [J]. Virol,1987,143:153-156.
[16]Terry G..M., Ho Terry L. M., Londesborough P.,et al. Localization of the rubella El epitopes[J]. Arch Virol,1988,98:189-197.
[17]Bosma TJ, Best JM, Corbett KM, et al. Nucleotide sequence analysis of a major antigenic domain of the E1 glycoprotein of 22 rubella virus isolates[J]. J Gen Virol,1996,77 (Pt 10): 2523-2530.
[18]Mitchell L.A., ZhangT., Ho M.et al. Characterization of Rubella specific antibody responses by using a new synthetic peptide basedenzyme linked immunosorbent assay[J]. J.Clin. Microbiology. 1992,30;1841-1847.
[19]Chaye H., Chong P., Tripet B. et al. Localization of the virus neutralizing and hemagglutinin epitopes of E1 glycoprotein of rubella virus[J]. Virology,1992,189:483-492.
[20]Chen M.J., Icenogle J.P., Molecular virology of rubella virus. Rubella Virus, J.Banatvala andC.Peekham[J].Oxford, UK, Elsevier,2007:1-18.
[21]Frey TK, Abernathy ES, Bosma TJ,et al. Molecular analysis of rubella virus epidemiology across three continents, North America, Europe, and Asia,1961-1977 [J]. J Infect Dis, 1998,178(3):642-650.
[22]Katow S, Minahara H, Fukushima M, et al. Molecular epidemiology of rubella by nucleotide sequences of the rubella virus El gene in three East Asian countries[J]. Infec Dis,1997,176(3):606-616.
[23]Best JM, Thomson A, Reynors J,et al. Rubella virus strains show no major antigenic differences[J]. Intervirology,1992,34(3):164-168.
[24]Waxham M.N, Wolinsky J.S.. A Model of the Structural Organization of Rubella Virions[J]. Clin Infect Dis,1985,7 (Suppl 1):133-139.
[25]Dalgarno L., Rice C.M., Sreauss J.H.. Ross River virus 26 s RNA:complete nucleotide sequence and deduced sequence of the encoded structural proteins. [J]. Virology,1983,129:170-187.
[26]Hobman TC, Woodward L, Farquhar MG. Targeting of a heterodimeric membrane protein complex to the Golgi:rubella virus E2 glycoprotein contains a transmembrane Golgi retention signal[J]. Mol Biol Cell,1995,6(1):7-20.
[27]Law LM, Duncan R, Esmaili A,et al. Rubella virus E2 singal petide is required for perinuclear localization of capsid protein and virus assembly[J]. J Virol,2001,75(4):1978-1983.
[28]Parkman PD. Making vaccination policy:the experience with Rubella[J]. Clin Infect Dis,1999,28(12):140-146.
[29]Qiu Z, Hobman TC, McDonald HL, et al. Role of N-linked oligosaccharides in processing and intracellular transport of E2 glycoprotein of Rubella virus[J]. Virology,1992,66(6):3514-3521.
[30]Terry H L.,Terry G.M., Londesborough P. Diagnosis of focusing and peptide mapping[J]. Arch Virol,1982,72:47-54.
[31]Dorsett PH, Miller DC, Green KY, et al. Structure and function of the rubella virus proteins [J]. Rev Infect Dis.,1985,7 (Suppl 1):150-156.
[32]Cusi MG, Metelli R, Valensin PE. Immune responses to wild and vaccine rubella viruses after rubella vaccination[J]. Arch Virol,1989,106(1-2):63-72.
[33]Green KY, Dorsett PH. Rubella virus antigens:localization of epitopes involved in hemagglutination and neutralization by using monoclonal antibodies[J]. J Virol,1986, 57(3):893-898.
[34]王志玉.风疹病毒包膜糖蛋白研究进展[J].国外医学·微生物学分册,1995,18(4)：12-13.
[35]Marr LD, Sanchez A, Frey TK. Efficient in vitro translation and proccssing of rubella virus structural proteins in the presence of microsomes[J]. Virology,1991,180(1):400-405.
[36]Bowden DS, Westaway EG. Rubella virus:structrual and non-structural proteins[J]. J Gen Virol,1984,65(5):933-943.
[37]Liu Z, Yang D, QiuZ, et al. Idnetification of domains in rubella viurs genomic RNA and cpasid protein necessary for specific interaction[J]. J Viorl,1996,70(4):2184-2190.
[38]Law LM, Everitt JC, Beatch MD, etal. Phosphorylation of rubella virus capsid regulates its RNA bingding activity and virus replication[J]. J Virol,2003,77(3):1764-1771.
[39]Tzeng WP, Frey TK.ComPlementation of a deletion in the rubella virus p150 nonstructural protein by theviral capsid protein[J]. J Virol,2003,77(17):9502-9510.
[40]Duncan R, Esmaili A, Law LM, et al. Rubella virus capsid protein induces apoptosis in transfected RK13 cells[J]. Virology,2000,275(1):20-29.
[41]Cusi M.G., Rossolini G.M., Cellesi C.,et al. Antibody response to wild rubella virus structural proteins following immunization witg RA27/3 live attenuated vaccine[J].Arch Virol,1988,101:25-33.
[42]彭文伟主编.现代感染性疾病与传染病学[M].第一版.北京：科学出版社.2000：642-653.
[43]Smith CA, Petty RE, Tingle AJ. Rubella virus and arthlitis[J]. Rheum Dis Clin North Am,1987,13(2):265-274.
[44]Portielje J.E.A., Westendorp R.G.J., Kluin-Nelemans H.C., et al. Morbidity and mortality in adults with idiopathic thrombocytopenic purpura[J]. Blood,2001,97 (9):2549-2554.
[45]Baron M.D., Ebel T., Suomalainen M.Inltracellular transport of rubella virus structural proteins expressed from cloned cDNA[J].JGenVirol,1992,73(5):1073-1086
[46]Ruokonen PC, Metzner S, Ucer A, et al. Intraocular antibody synthesis against rubella virus and other microorganisms in Fuchs' heterochromic cyclitis[J]. Graefes Arch Clin Exp Ophthalmol,2010,248:565-571
[47]Bossy-Wetztel E, Green DR. Apoptosis:check point at the mitochondrial frontier[J].Mutat Res,1999,434(3):243-251.
[48]Robertson S.E., Featherstone D.A., Gacic-Dodo M.,et al. Rubella and congeni-tal rubella syndrome:global update[J]. Rev Panam Salud Publica,2003,14(5):306-315.
[49]Cutts, F.T., Vynnyck E..Modelling the incidence of congenital rubella sydrome in developing countries[J]. Int J Epidemiol,1999,28(6):1176-1184.
[50]Lee J., Bowden D.S..Rubella virus replication and links to teratogenicity[J].Clin Microbiol Rev, 2000,13(4):571-587.
[51]Reece EA, Hobbin JC. Medicine of the fetus&mother,2nded[M]. Philadephia:Lippincott-Raven New York,1999:347-365.
[52]李沪,胡家瑜,陶黎纳,等.先天性风疹综合征流行病学特征与免疫预防策略[J].上海预防医学杂志,2005,17(2)：72-74.
[53]KurodaY, Matsui M. Progressive rubella panencephalitis[J]. Nippon Rinsho,1997, 55(4):922-925.
[54]Webster WS. Teratogen update:congenital rubella[J]. Teratology,1998,58(1):13-23.
[55]Miller E, Cradoek-Wstson JE, Pollock TM. Consequences of confirmed maternal rubella at successive stages of pregnancy [J]. Lancet,1982,302(82):781-784.
[56]Megyeri K, Berenesi K, Halazonetis TD, et al.Involvement of a P53-dependent pathway in rubella virus-induced apoptosis[J]. Viorlogy,1999,259(1):74-84.
[57]Hofmann J, Pletz MW, Liebert UG. Rubella virus-induced cytopathic effect in vitro is caused by apoptosis[J]. J Gen Virol,1999,80(7):1657-1664.
[58]Novitski E. Human Genetics P34[M]. Macmillam publishing Co Inc.1997.
[59]Kono R, Hirayama M, Sugishita C, et al. Epidemiology of Rubella and Congenital Rubella Infection in Japan[J]. Reviews of Infectious Diseases,1985,7:56-63
[60]天津医学院流行病教研室.人类病毒性传染病·流行病学及防治[M].北京：人民卫生出版社,1984：442.
[61]Reef S.E. Redd SB, Abernathy E, et al. The epidemiological profile of rubella and congenital rubella syndromein the United States,1998-2004:the evidence for absence of endemic transmission[J].Clin Infect Dis,2006,43,Suppl 3:126-132.
[62]Icenogle, J.P., Frey TK, Abernathy E, et al..Genetic analysis of rubella viruses found in the United States between 1966 and 2004:evidence that indigenous rubella viruses have been eliminated[J]. Clin Infect Dis,2006,43Suppl 3:133-140.
[63]CDC, Elimination of rubella and congenital rubella syndrome-United Srates, 1969-2004[J].MMWR Morb Mortal Wkly Rep,2005,54(11):279-282.
[64]CDC, Achievements in public health:elimination of rubella and congenital rubella syndrome-US,1969-2004[J]. Ann Pharmacother,2005,39(6):1151-1152.
[65]WHO. Progress towards eliminating rubella and congenital rubella syndrome in the western hemisphere,2003-2008. Wkly Epidemiol Rec,2008,83:395-400.
[66]Peltola H, Jokinen S, Paunio M, et al. Measles, mumps, and rubella in Finland:25 years of a nationwide elimination programme[J]. Lancet Infect Dis.2008,8(12):796-803.
[67]D'Agaro P, Dal Molin G, Zamparo E, et al. Epidemiological and molecular assessment of a rubella outbreak in North-Eastern Italy[J]. J Med Virol.2010,82(11):1976-1982.
[68]Cutts FT, Robertson SE, Samuel R, et al. Control of rubella and congenital rubella syndrome(CRS)in developing countries, part 1:burden of disease from CRS[J]. Bull World Health Organ,1997,75(1):55-68.
[69]樊春祥,梁晓峰,周玉清,等.中国2004-2006年风疹流行病学特征初步分析[J].中国计划免疫,2007,13(5)：457-460.
[70]朱贞,郭学斌,崔爱利,等.中国2007-2008年风疹流行病学和病毒基因特征分析[J].中国疫苗和免疫,2009,15(3)：201-204.
[71]马静,郝利新,罗会明,等.中国2008-2009年风疹流行病学特征分析[J].中国疫苗和免疫,2010,16(4)：322-324.
[72]周先军.1082例妊娠妇女TORCH感染情况调查分析[J].检验医学与临床,2010,7(8)：680-681.
[73]陈世勇,赵真真,阮林松,等.早孕妇女1379例TORCH感染情况分析[J].实用医学杂志,2010,26(4)：708-709.
[74]王丽娟,王志玉.风疹病毒检测方法研究进展[J].中国公共卫生,2008,24(6)：749-751.
[75]Gurtadauria S, Cordoba P, Cuffini C, et al. Cell-fusion assay for the detection of rubella virus in Vero cells[J]. Clin Diagn Virol,1998,10(1):9-16.
[76]Yan H, Li G, Yan H,et al. Establishment and characterization of a novel genetic hybrid cell line for propagation of four pathogens [J]. J Virol Methods,2006,134 (1-2):86-91.
[77]Christopher Mpale PA, Jones CS. Time-resolved fluoromertic irnrnunoassay for rubella antibody-a useful method for serosurveillance studies[J]. Vaccine,2002,20 (9-10):1378-1382.
[78]朱贞,许文波,李聪勇,等.一种新型诊断方法—比色法免疫学实验在检测风疹病毒中的应用研究[J].中国计划免疫,2006,12(6)：489-493.
[79]Perry KR, Brown DW, Parry JV, et al. Detection of measles, mumps, and rubella antibodies in saliva using antibody capture radioimmunoassay [J]. J Med Virol,1993,40(3):235-240.
[80]Voller A, Bidwell DE. A simple method for detecting antibodies to rubella[J]. Br J Exp Pathol, 1975,56(4):338-339.
[81]Morris M, Cohen B,Andrews N,et al. Stability of total and rubella-specific IgG in oral fluid samples:the effect of time and temperature[J]. J Immunol Methods,2002,266 (1-2):111-116.
[82]Akingbade D, Cohen BJ, Brown DW. Detection of low-avidity immunoglobulin G in oral fluid samples:new approach for rubella diagnosis and surveillance [J]. Clin Diagn Lab Immunol,2003,10(1):189-190.
[83]Gay NJ, Vyse AJ, Enquselassie F, et al.Implroving sensitivity of oral fiuid testing in IgG pervalence studies:application of mixture models to a rubella antibody survey[J]. Epidemiol Infect,2003,130(2):285-291.
[84]Allmendinger J., Paradies F., Kamprad M., et al. Determination of Rubella Virus-Specific Cell-Mediated Immunity Using IFNy-ELISpot[J]. Journal of Medical Virology,2010,82:335-340.
[85]Ardizzoni A, Capuccini B, Baschieri MC, et al. A protein microarray immunoassay for the serological evaluation of the antibody response in vertically transmitted infections[J]. Eur J Clin Microbiol Infect Dis.2009,28(9):1067-1075
[86]Li D, Yang H, Zhang WH, et al. A simple parallel analytical method of prenatal screening[J]. Gynecol Obstet Invest,2006,62 (4):220-225.
[87]Tanemura M, Suzmuori K, Yagami Y,et al. Diagnosis of fetal rubella infection with reverse transcription and nested polymerase chain reaction:a study of 34 cases diagnosed in fetuses[J]. Am J Obstet Gynecol,1996,174(2):578-582.
[88]Vyse AJ, Jin L.An RT-PCR assay using oral fluid samples to detect rubella viurs genome for epidemiological surveillnace[J].Mol Cell Probes,2002,16(2):93-97.
[89]Shyamala G,Malathi J,Moses YS,et al. Nested reverse transcription polymerase chain reaction for the detection of rubella virus in clinical specimens[J]. Indian J Med Res,2007,125 (1):73-78.
[90]Cooray S,Warrener L,Jin L. Improved RT-PCR for diagnosis and epidemiological surveillance of rubella[J]. J Clin Virol,2006,35 (1):73-80.
[91]Mosuqera MM, Ory FD,Moreno M,et al.Simultaneous detection of measles viurs,rubella viurs, and parvovirus by using multiplex PCR[J].J Clin Micorbiol,2002.40(1):111-116.
[92]Feng Y, Santibanez S, Appleton H, et al. Application of new assays for rapid confirmation and genotyping of isolates of rubella virus[J]. J Med Virol.2011,83(1):170-177.
[93]Mosquera M M,Sanz JC, Echevarria J E,et al. Diagnostic performance of specific IgM detection and genomic amplification in rubella [J]. Enferm Infect Microbiol Clin,2006,24 (4): 251-253.
[94]Liu Q,Bai Y, Ge Q,et al. Microarray-in-a-tube for detection of multiple viruses[J]. Clin Chem,2007,53(2):188-194.
[95]Maeda H, Kokeguchi S, Fujimoto C, et al. Detection of periodontal pathogen Porphyromonas gingivalis by loop-mediated isothermal amplification method [J]. FEMS Immunol Med Microbiol, 2005,43:233-239.
[96]Nagamine K, Hase T,Notomi T. Accelerated reaction by loop-mediated isothermal amplification using loop primers[J]. Molecular and Cellular Probes,2002,16 (3):223-229.
[97]Mori N,Motegi Y, Shimamura Y, et al. Development of a new method for diagnosis of rubella virus infection by reverse transcription-loop-mediated isothermal amplification[J]. J Clin Microbiol,2006,44 (9):3268-3273.
[98]Song T, Toma C, Nakasone N, et al. Sensitive and rapid detection of Shigella and enteroinvasive Escherichia coli by a loop-mediatied isothermal amplification method [J]. FEMS Microbiol Lett,2005,243:259-263.
[99]胡得意,胡永卫,李晓祺,等.学生风疹发病危险因素的病例对照研究[J].浙江预防医学,2010,22(2)：20.
[100]马岚,曹文正,史红,等.兰州市2005-2008年麻疹、风疹实验室检测结果分析[J].中国初级卫生保健,2010,24(2)：76-77.
[101]Prodromou C. Recursive PCR:a novel technique for total gene synthesis [J].Protein Engineering,1992,5(8):827-829.
[102]王志玉,薛永磊,王小凡,等.风疹病毒J R23株E1包膜糖蛋白基因的表达分析[J].病毒学报,2003,19(3)：278-280.
[103]温红玲,王志玉,宋艳艳,等.风疹病毒J R23株糖蛋白E1在毕赤酵母表达系统中的表达和抗原性分析[J].中华微生物学和免疫学杂志,2005,25(3)：190-193.
[104]Hobman TC, Lundstrom ML, Gillam S. Processing and intracellular transport of rubella virus strucural proteins on COS cells. Virology,1990,178:122-133.
[105]Oker-Blom C, Pettersson RF, Summers MD. Baculovirus polyhedron promoterdirected expression of rubella virus envelope glycoproeins E1 and E2, in spodoptera frugiperda cells[J]. Virology,1989,172:82-91.
[106]Oiu ZY, Frank T, Shirley G. The influence of N-linked glycosylation on the antigenicity of rubella virus E1 glycoprotein[J].Virology,1992,190(2):876-881.
[107]Terry HL, Cohen A. The role of glycosylation on haemagglutination and immunological reactivity of rubella virus[J]. Arch Virol,1984,79:139-146.
[108]朱贵明,陈宏,卢建申,等.应用PCR-酶切连接法合成全长sFatl基因[J].遗传,2006,28(9)：1123-8.
[109]Terry GM, Terry HL, Lonesborough P., et al. A bio-engineered rubella E1 antigen [J]. Arch Virol,1989,104 (1-2):63-75.
[110]Starkey WG, Newcombe J, Corbett KM, et al. Use of rubella virus E1 fusion proteins for detection of rubella virus antibodies[J]. J Clin Microbiol,1995,33(2):270-274.