抗流感病毒B单抗杂交瘤细胞cDNA文库的构建及生物信息学分析
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摘要
流行性感冒(influenza,简称流感)是由流行性感冒病毒引起的急性呼吸道传染病,是人类面临的主要公共健康问题之一。据统计,流感每年的发病率为10%～30%,其流行病学的特点是:突然暴发,迅速蔓延,播及面广。流感流行具有一定的季节性。我国北方常发生于冬季,而南方多发生在冬夏两季。流感发病率高,人群普遍易感。流感临床症状较重,起病急骤,并发症发生率高,特别是肺炎,可引起死亡,死者大多为年迈体弱和幼年多病或有慢性基础病者。B型流感病毒近年来常造成流感局部区域性暴发。
病毒的感染过程其实就是病毒与宿主或机体相互斗争的过程。杂交瘤细胞是一种“1+1>2”的特殊细胞,其分泌抗体的基因源于受到免疫原刺激的宿主免疫细胞――B细胞。她的基因组承载着病原体与宿主相互作用的信息,同时由于整合了肿瘤细胞的“长生基因”,使其成活时间明显延长,而且极有可能产生一些基因的突变和重组,并由此产生一些抗体之外的新物质。因此流感病毒B单抗杂交瘤细胞基因组的研究是一个值得探索的课题。如何迅速、高效的从基因组中获取生物学信息,是一个富有挑战性的课题,EST技术就是基于这种认识而发展起来的。
本试验用纯化的Flu B抗原免疫小鼠,拟制备能稳定分泌特异性单克隆抗体的杂交瘤细胞株,为研制一种方便、快捷、特异的金标试纸快速诊断方法打下坚实的基础,同时拟构建分泌抗Flu B抗体杂交瘤细胞cDNA文库并测序,获取EST片断,再通过美国国家生物信息中心(National Center Biotechnology Information, NCBI)服务器的UniGene数据库进行BLAST检索,应用sequencher软件对EST序列进行片断重叠群分析,应用生物信息学方法研究杂交瘤基因,为预测和进一步开发杂交瘤细胞的功能提供理论基础。
方法和结果:
1.应用纯化的B型流感病毒抗原免疫BALB/c小鼠,经过5次免疫、细胞融合、融合细胞的亚克隆,获得了3株能稳定分泌抗B型流感病毒单克隆抗体的杂交瘤细胞,分别命名为:3B2,12G3,5A4。3株杂交瘤细胞株染色体计数检测显示接近亲本细胞染色体数目之和。3株单抗的重链均为IgG1型,轻链为κ链,单抗效价较高、与A型流感病毒和RSV均不发生交叉反应。
2.应用经典cDNA文库构建方法,经过细胞总RNA的提取、mRNA的分离,以带有多聚腺苷酸的mRNA为模板,反转录合成cDNA第一链,第二链,cDNA双链和载体的连接,转入受体菌,构建成杂交瘤细胞的cDNA文库。该文库库容为1.27×106。
3.原始文库中挑选了400个克隆,测序共得到384个序列,测序成功率为96%,将序列编辑后去除载体序列后得到长度在200bp～700bp的有效序列320条。经过BLAST程序对所获得的EST片断进行基因,结果显示有118条序列与数据库中的已知基因有较好的相似性。经过Sequencher 4.8软件将384条EST片断的进行片断重叠群(contig)分析,结果共得到包括71条EST序列在内的25组片断重叠群,占全部EST序列的22.2%,其余249条都属于单拷贝序列。
结论:
1.成功制备抗B型流感病毒杂交瘤细胞株,为金标法快速诊断B型流感病毒感染奠定了基础。
2.抗B型流感病毒杂交瘤细胞cDNA文库的成功构建和部分克隆的测序,对于新基因的克隆和功能研究提供了可用资源,同时对预测和进一步开发杂交瘤细胞的功能提供了良好的物质基础。
3.为建立杂交瘤细胞的资源信息库做了重要的开端工作。
Influenza caused by influenza virus was a acute respiratory tract infectious disease, spread rapidly and easily antigen variation. Virus′s infection process is actually the process that mutual struggle process of virus and the host or organism. hybridoma cell was a special composite cell,whose secretory antibody gene was originated B cell which was a immunocyte irritated by immunogen, B cell′s genome bore the weight of interactive information between infector and parasitifer, meanwhile, integrated with macrobiotic gene originated by tumour cell, so hybridoma cell had a relative survival time, maybe mutation and recombination were generated, then produce material besides antibody. Researching of genome of hybridoma cell was a Subject worth exploring. How to obtain biological information fast and efficiently from the genome has become an urgent and challenging task, EST technology was just developed based on this understanding.
Influenza B virus was used for the immunization of BALB/c mice. The hybridoma cell strains stably secreting McAbs against Influenza B virus were obtained by fusion of murine splenocytes with SP2/0 plasmacytoma cells, and the secreted McAb was identified. The hybridoma cell was cultured in vitro. Total RNA of hybridoma cell was extracted from the cultured cells and then mRNA was extracted further. Moreover ,single - strand cDNA and double - strand cDNA were synthesized in turn. The double - strand cDNAs were ligated to EcoRI adaptor ,which were later ligated to arms of pBluescriptⅡSK(+) XR.Ligated - cDNAs were packed in vitro, then the hybridoma cell cDNA library was constructed. Sequence of EST was obtained by sequencing random, then had BLAST index through Unigene Server of National Center Biotechnology Information(NCBI). The sequencher software was used to set up and analysis EST contig. In order to establish the foundations that studing gene and predict or exploitation function of hybridoma cell.
The results show that: 1) Three hybridoma cell strains which can steadily secrete specific McAbs were obtained, named 3B2,12G3,5A4. chromosome number of hybridoma approach that of parent cell, McAb heavy chain belonged to IgG1, and light chain belonged to Kappa. titer of McAb secreted by three strains was higher ennogh. The specificity identify showed that McAb did not react with influenza A and RSV. 2) The hybridoma cell cDNA library consisted of 1.27×106 recombinants with the length of 0.5～2.0kb and the recombinating efficiency was 90.63% . The amplified library was 3 .5×107 recombinants/μl in concentration and the number of negative colony was the most suitable in density after it was diluted to 10 - 6 in concentration. 3) 400 clones were selected randomly from archaelibrary to sequence, 384 sequences were obtained from the plamids, and 320 ESTs were proved to be useful after edited using biosoftware. After analysis by using the BLAST program, 118 ESTs were proved to be the known-genes, and ESTs whose aligment value were low maybe unknown-genes. Contig analisis of 320 ESTs by Sequencher 4.8 showed that 25 comtig including 71 ESTs which was 22.2% in all EST were obtained. The rest EST were single-copy sequence.
Conclusion: 1) The McAbs established by our method could be highly specific for Influenza B virus, which provides a basic condition for development of rapid diagnosis method for Influenza B virus. 2) The constructed cDNA library of hybridoma cell and sequencing of partial clones provided useful resource to study function of new genes, further detecting target genes, at the same time, to predict or exploit function of hybridoma cel. 3)Accomplishment of this thesis is a important initial work for building resource information databank of hybridoma cell.
病毒的感染过程其实就是病毒与宿主或机体相互斗争的过程。杂交瘤细胞是一种“1+1>2”的特殊细胞,其分泌抗体的基因源于受到免疫原刺激的宿主免疫细胞――B细胞。她的基因组承载着病原体与宿主相互作用的信息,同时由于整合了肿瘤细胞的“长生基因”,使其成活时间明显延长,而且极有可能产生一些基因的突变和重组,并由此产生一些抗体之外的新物质。因此流感病毒B单抗杂交瘤细胞基因组的研究是一个值得探索的课题。如何迅速、高效的从基因组中获取生物学信息,是一个富有挑战性的课题,EST技术就是基于这种认识而发展起来的。
本试验用纯化的Flu B抗原免疫小鼠,拟制备能稳定分泌特异性单克隆抗体的杂交瘤细胞株,为研制一种方便、快捷、特异的金标试纸快速诊断方法打下坚实的基础,同时拟构建分泌抗Flu B抗体杂交瘤细胞cDNA文库并测序,获取EST片断,再通过美国国家生物信息中心(National Center Biotechnology Information, NCBI)服务器的UniGene数据库进行BLAST检索,应用sequencher软件对EST序列进行片断重叠群分析,应用生物信息学方法研究杂交瘤基因,为预测和进一步开发杂交瘤细胞的功能提供理论基础。
方法和结果:
1.应用纯化的B型流感病毒抗原免疫BALB/c小鼠,经过5次免疫、细胞融合、融合细胞的亚克隆,获得了3株能稳定分泌抗B型流感病毒单克隆抗体的杂交瘤细胞,分别命名为:3B2,12G3,5A4。3株杂交瘤细胞株染色体计数检测显示接近亲本细胞染色体数目之和。3株单抗的重链均为IgG1型,轻链为κ链,单抗效价较高、与A型流感病毒和RSV均不发生交叉反应。
2.应用经典cDNA文库构建方法,经过细胞总RNA的提取、mRNA的分离,以带有多聚腺苷酸的mRNA为模板,反转录合成cDNA第一链,第二链,cDNA双链和载体的连接,转入受体菌,构建成杂交瘤细胞的cDNA文库。该文库库容为1.27×106。
3.原始文库中挑选了400个克隆,测序共得到384个序列,测序成功率为96%,将序列编辑后去除载体序列后得到长度在200bp～700bp的有效序列320条。经过BLAST程序对所获得的EST片断进行基因,结果显示有118条序列与数据库中的已知基因有较好的相似性。经过Sequencher 4.8软件将384条EST片断的进行片断重叠群(contig)分析,结果共得到包括71条EST序列在内的25组片断重叠群,占全部EST序列的22.2%,其余249条都属于单拷贝序列。
结论:
1.成功制备抗B型流感病毒杂交瘤细胞株,为金标法快速诊断B型流感病毒感染奠定了基础。
2.抗B型流感病毒杂交瘤细胞cDNA文库的成功构建和部分克隆的测序,对于新基因的克隆和功能研究提供了可用资源,同时对预测和进一步开发杂交瘤细胞的功能提供了良好的物质基础。
3.为建立杂交瘤细胞的资源信息库做了重要的开端工作。
Influenza caused by influenza virus was a acute respiratory tract infectious disease, spread rapidly and easily antigen variation. Virus′s infection process is actually the process that mutual struggle process of virus and the host or organism. hybridoma cell was a special composite cell,whose secretory antibody gene was originated B cell which was a immunocyte irritated by immunogen, B cell′s genome bore the weight of interactive information between infector and parasitifer, meanwhile, integrated with macrobiotic gene originated by tumour cell, so hybridoma cell had a relative survival time, maybe mutation and recombination were generated, then produce material besides antibody. Researching of genome of hybridoma cell was a Subject worth exploring. How to obtain biological information fast and efficiently from the genome has become an urgent and challenging task, EST technology was just developed based on this understanding.
Influenza B virus was used for the immunization of BALB/c mice. The hybridoma cell strains stably secreting McAbs against Influenza B virus were obtained by fusion of murine splenocytes with SP2/0 plasmacytoma cells, and the secreted McAb was identified. The hybridoma cell was cultured in vitro. Total RNA of hybridoma cell was extracted from the cultured cells and then mRNA was extracted further. Moreover ,single - strand cDNA and double - strand cDNA were synthesized in turn. The double - strand cDNAs were ligated to EcoRI adaptor ,which were later ligated to arms of pBluescriptⅡSK(+) XR.Ligated - cDNAs were packed in vitro, then the hybridoma cell cDNA library was constructed. Sequence of EST was obtained by sequencing random, then had BLAST index through Unigene Server of National Center Biotechnology Information(NCBI). The sequencher software was used to set up and analysis EST contig. In order to establish the foundations that studing gene and predict or exploitation function of hybridoma cell.
The results show that: 1) Three hybridoma cell strains which can steadily secrete specific McAbs were obtained, named 3B2,12G3,5A4. chromosome number of hybridoma approach that of parent cell, McAb heavy chain belonged to IgG1, and light chain belonged to Kappa. titer of McAb secreted by three strains was higher ennogh. The specificity identify showed that McAb did not react with influenza A and RSV. 2) The hybridoma cell cDNA library consisted of 1.27×106 recombinants with the length of 0.5～2.0kb and the recombinating efficiency was 90.63% . The amplified library was 3 .5×107 recombinants/μl in concentration and the number of negative colony was the most suitable in density after it was diluted to 10 - 6 in concentration. 3) 400 clones were selected randomly from archaelibrary to sequence, 384 sequences were obtained from the plamids, and 320 ESTs were proved to be useful after edited using biosoftware. After analysis by using the BLAST program, 118 ESTs were proved to be the known-genes, and ESTs whose aligment value were low maybe unknown-genes. Contig analisis of 320 ESTs by Sequencher 4.8 showed that 25 comtig including 71 ESTs which was 22.2% in all EST were obtained. The rest EST were single-copy sequence.
Conclusion: 1) The McAbs established by our method could be highly specific for Influenza B virus, which provides a basic condition for development of rapid diagnosis method for Influenza B virus. 2) The constructed cDNA library of hybridoma cell and sequencing of partial clones provided useful resource to study function of new genes, further detecting target genes, at the same time, to predict or exploit function of hybridoma cel. 3)Accomplishment of this thesis is a important initial work for building resource information databank of hybridoma cell.
引文
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1. Neumann G,Whitt MA,Kawaoka Y. A decade after the generation of a negative-sense RNA virus from cloned cDNA—What have we learned?J Virol,2002,83:2635-2662.
2. Lutjes W,Krystal M,Enami M,et al. Amplification,expression and packaging of foreign gene by influenza virus. Cell,1989,59(6):1107-1113.
3. Neumann G,Watanabe T,Ito H,et al. Generation of influenza A virus entirely from cloned cDNAs. Proc Natl Acod Sci U S A,1999,96(16):9345-9350.
4. Hoffmann E,Neumann G,Hobom G,et al.“Ambisense”approach for the generation of influenza A virus: vRNA and mRNA synthesis from one template. Virology,2000,267:310-317.
5.陈稚峰,张立国,董婕等。应用反向遗传学技术在哺乳动物细胞中产生甲型流感病毒。病毒学报,2002,18(3):193-197。
6. Schnell MJ,Mebatsion T,Conzelmann KK. Infection rabies viruses from cloned cDNA . EMBO J,1994,13(18):4195-4103.
7. Collins PL,Hill MG,Camargo E,et al. Production of infectious human respiratory syncytial virus from cloned cDNA confirms an essential role for the transcription elongation factor from the 5′proximal open reading frame of the M2 mRNA in gene expression and provides a capability for vaccine development. Proc Natl Acod Sci U S A,1995,92(25):11563-11567.
8. Hoffman MA,Banerjee AK. An infectious clone of human parainfluenza virus type 3. J Virol,1997,71(6)4272-4277.
9. Durbin AP,Hall SL,Siew JW,et al. Recovery of infectious human parainfluenza virus type 3 from cloned cDNA . Virology,1997,235(2):323-332.
10. Taubenberger JK,Reid AH,Bijwaard KE,et al. Initial genetic characterization of the 1918“spanish”influenza virus. Science,1997,275:1793-1795.
11. Reid AH,Fanning TG,Hμltin JV,et al. Origin and evolution of the 1918 'Spanish' influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999,96:1651–1656.
12. Kobasa D,Takada A,Shinya K,et al. Enhanced virμlence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature,2004,431:703-707.
13. Basler CF,Reid AH,Dybing JK,et al. Sequence of the 1918 pandemic influenza virus nonstructural gene(NS)segment and characterization of recombinant virus bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001,98(5):2746-2751.
14. Reid AH,Fanning TG,Janczewski TA,et al. Characterization of the 1918 'Spanish' influenza virus matrix gene segment. J Virol,2002,76(21):10717–10723.
15. Reid AH,Fanning TG,Janczewski TA,et al. Novel Origin of the 1918 Pandemic Influenza Virus Nucleoprotein Gene. J Virol,2004,78(22):12462– 12470.
16. Taubenberger JK,Reid AH,Lourens RM,et al. Characterization of the 1918 influenza virus polymerase genes. Nature,2005,437:889-893.
17. Tumpet TM,Baster CF,Aguilar PV,et al. Characterization of the reconstructed 1918 spanish influenza pandemic virus. Science,2005,301:77-80.
18. Zimmer G,Conzelmann KK,Herrler G. Cleavage at the furin consensus sequence RAR/KR(109) and presence of the intervening peptide of the respiratory syncytial virus fusion protein are dispensable for virus replication in cell cμlture. J Virol,2002,76(18):9218-9224.
19. Hoffmann E,Krauss S,Perez D,et al. Eight-plasmid system for rapid generation of influenza virus vaccines. Vaccine,2002,20:3165-3170.
20. Webby RJ,Perez DR,Coleman JS,et al. Responsiveness to a pandemic alert:use of reverse genetics for rapid development of influenza vaccines. Lancet,2004,363:1099-1103.
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